Isolation of binding proteins with high affinity to ligands

ABSTRACT

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via “display-less” library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as  Escherichia coli , and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.

This application is a divisional application of currently applicationSer. No. 09/699,023, filed Oct. 27, 2000 now U.S. Pat. No. 7,083,945,the contents of which are incorporated herein by reference.

The government owns rights in the invention pursuant to DARPA Grant No.MDA(972-97-0009).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of proteinengineering. More particularly, it concerns methods for the screening ofcombinatorial libraries of polypeptides to allow isolation of enzymeshaving a desired catalytic activity and of ligand binding proteins,including antibodies and binding proteins having affinity for selectedligands

2. Description of Related Art

The isolation of proteins that either bind to ligands with high affinityand specificity or catalyze the enzymatic conversion of a reactant(substrate) into a desired product is a key process in biotechnology.Ligand-binding proteins and enzymes with a desired substrate specificitycan be isolated from large libraries of mutants, provided that asuitable screening method is available. Small protein libraries composedof 10³-10⁵ distinct mutants can be screened by first growing each cloneseparately and then using a conventional assay for detecting clones thatexhibit specific binding. For example, individual clones expressingdifferent protein mutants can be grown in microtiter well plates orseparate colonies on semisolid media such as agar plates. To detectbinding the cells are lysed to release the proteins and the lysates aretransferred to nylon filters, which are then probed using radiolabeledor fluorescently labeled ligands (DeWildt et al. 2000). However, evenwith robotic automation and digital image systems for detecting bindingin high density arrays, it is not feasible to screen large librariesconsisting of tens of millions or billions of clones. The screening oflibraries of that size is required for the de novo isolation of enzymesor protein binders that have affinities in the nanomolar range.

The screening of very large protein libraries has been accomplished by avariety of techniques that rely on the display of proteins on thesurface of viruses or cells (Ladner et al. 1993). The underlying premiseof display technologies is that proteins engineered to be anchored onthe external surface of biological particles (i.e., cells or viruses)are directly accessible for binding to ligands without the need forlysing the cells. Viruses or cells displaying proteins with affinity fora ligand can be isolated in a variety of ways including sequentialadsorption/desorption form immobilized ligand, by magnetic separationsor by flow cytometry (Ladner et al. 1993, U.S. Pat. No. 5,223,409,Ladner et al. 1998, U.S. Pat. No. 5,837,500, Georgiou et al. 1997,Shusta et al. 1999). The most widely used display technology for proteinlibrary screening applications is phage display. Phage display is awell-established and powerful technique for the discovery of proteinsthat bind to specific ligands and for the engineering of bindingaffinity and specificity (Rodi and Makowski, 1999). In phage display, agene of interest is fused in-frame to phage genes encodingsurface-exposed proteins, most commonly pill. The gene fusions aretranslated into chimeric proteins in which the two domains foldindependently. Phage displaying a protein with binding affinity for aligand can be readily enriched by selective adsorption onto immobilizedligand, a process known as “panning”. The bound phage is desorbed fromthe surface, usually by acid elution, and amplified through infection ofE. coli cells. Usually, 4-6 rounds of panning and amplification aresufficient to select for phage displaying specific polypeptides, evenfrom very large libraries with diversities up to 10¹⁰ Several variationsof phage display for the rapid enrichment of clones displaying tightlybinding polypeptides have been developed (Duenas and Borrebaeck, 1994;Malmborg et al., 1996; Kjaer et al., 1998; Burioni et al., 1998;Levitan, 1998; Mutuberria et al., 1999; Johns et al., 2000).

One of the most significant applications of phage display technology hasbeen the isolation of high affinity antibodies (Dall'Acqua and Carter,1998; Hudson et al., 1998; Hoogenboom et al., 1998; Maynard andGeorgiou, 2000). Very large and structurally diverse libraries of scFvor F_(AB) fragments have been constructed and have been usedsuccessfully for the in vitro isolation of antibodies to a multitude ofboth synthetic and natural antigens (Griffiths et al., 1994; Vaughan etal., 1996; Sheets et al., 1998; Pini et al., 1998; de Haard et al.,1999; Knappik et al., 2000; Sblattero and Bradbury, 2000). Antibodyfragments with improved affinity or specificity can be isolated fromlibraries in which a chosen antibody had been subjected to mutagenesisof either the CDRs or of the entire gene CDRs (Hawkins et al., 1992; Lowet al., 1996; Thompson et al., 1996; Chowdhury and Pastan, 1999).Finally, the expression characteristics of scFv, notorious for theirpoor solubility, have also been improved by phage display of mutantlibraries (Deng et al., 1994; Coiaetal., 1997).

However, several spectacular successes notwithstanding, the screening ofphage-displayed libraries can be complicated by a number of factors.First, phage display imposes minimal selection for proper expression inbacteria by virtue of the low expression levels of antibody fragmentgene III fusion necessary to allow phage assembly and yet sustain cellgrowth (Krebber et al., 1996, 1997). As a result, the clones isolatedafter several rounds of panning are frequently difficult to produce on apreparative scale in E. coli. Second, although phage displayed proteinsmay bind a ligand, in some cases their un-fused soluble counterparts maynot (Griep et al., 1999). Third, the isolation of ligand-bindingproteins and more specifically antibodies having high binding affinitiescan be complicated by avidity effects by virtue of the need for gene IIIprotein to be present at around 5 copies per virion to complete phageassembly. Even with systems that result in predominantly monovalentprotein display, there is nearly always a small fraction of clones thatcontain multiple copies of the protein. Such clones bind to theimmobilized surface more tightly and are enriched relative to monovalentphage with higher affinities (Deng et al., 1995; MacKenzie et al., 1996,1998). Fourth, theoretical analysis aside (Levitan, 1998), panning isstill a “black box” process in that the effects of experimentalconditions, for example the stringency of washing steps to remove weaklyor non-specifically bound phage, can only be determined by trial anderror based on the final outcome of the experiment. Finally, even thoughpIII and to a lesser extent the other proteins of the phage coat aregenerally tolerant to the fusion of heterologous polypeptides, the needto be incorporated into the phage biogenesis process imposes biologicalconstraints that can limit library diversity. Therefore, there is agreat need in the art for techniques capable of overcoming theselimitations.

Protein libraries have also been displayed on the surface of bacteria,fungi, or higher cells. Cell displayed libraries are typically screenedby flow cytometry (Georgiou et al. 1997, Daugherty et al. 2000).However, just as in phage display, the protein has to be engineered forexpression on the cell surface. This imposes several potentiallimitations. First of all, either the N-terminal or the C-terminal ofthe protein has to be fused to a vehicle for display. Thus, thesetechnologies are not suitable where the N- or C-termini are essentialfor ligand binding. Second, the requirement for display of the proteinon the surface of a cell imposes biological constraints that limit thediversity of the proteins and protein mutants that can be screened.Third, complex proteins consisting of several polypeptide chains cannotbe readily displayed on the surface of bacteria, filamentous phages oryeast. As such, there is a great need in the art for technology whichcircumvents all the above limitations and provides an entirety novelmeans for the screening of very large polypeptide libraries.

At present, the isolation of novel enzymes from libraries of proteinmutants is typically accomplished either through the use of a phenotypicselection or screening in either solid phase or microtiter well plates.Biological selections are based on complementation of auxotrophy orresistance to cytotoxic agents (e.g., antibiotics). Unfortunately, theutility of phenotypic selections is limited to the isolation ofcatalysts for reactions that are of direct biological relevance or canbe indirectly linked to a selectable phenotype. Alternatively, eachclone in a mutant population may be screened directly for enzymaticactivity. For libraries expressed in microorganisms, screening can beperformed on colonies growing on a solid substrate such as agar. Solidphase screening relies on substrates of an enzymatic reaction that giverise to a zone of clearance, a fluorescent product, or a stronglyabsorbing (chromogenic) product. The assay may detect the enzyme productdirectly or may be coupled to a second enzyme whose product can in turnbe easily monitored. However, many assays cannot be implemented in asolid phase format. If that is the case then individual clones must begrown and assayed in microtiter wells. Such assays are significantlymore time consuming than solid phase assays and severely limit thenumber of mutants that can be screened. However, when a small number ofrandom mutants is screened, the probability of finding clones expressingan enzyme that can catalyze a desired biotransformation, especially whenthat biotransformation requires a complicated reaction, is severelyaffected.

In general, methods that will allow the screening of large libraries ofenzyme mutants on the basis of kinetic parameters, i.e., on the basis ofhow much product is generated per unit time, are needed. Phage displaytechnology may in principle be used as a tool for the isolation ofuseful enzymes from large libraries. However, harnessing phage displaytechnology for the isolation of enzyme catalysts from libraries has thusfar not proven practical (Olsen et al. 2000). For example there is noapparent way to physically link in a quantitative manner a phageparticle displaying a certain enzyme clone with the outcome of multiplecatalytic turnovers resulting in the accumulation of reaction product.Establishing such a linkage is essential for the screening of proteinlibraries on the basis of catalytic proficiency.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of obtaining a bacteriumcomprising a nucleic acid sequence encoding a binding protein capable ofbinding a target ligand, the method comprising the steps of: (a)providing a Gram negative bacterium comprising a nucleic acid sequenceencoding a candidate binding protein, wherein the binding protein isexpressed in soluble form in the bacterium; (b) contacting the bacteriumwith a labeled ligand capable of diffusing into the bacterium; and (c)selecting the bacterium based on the presence of the labeled ligandwithin the bacterium, wherein the ligand and the candidate bindingprotein are bound in the bacterium.

In another aspect, the invention provides a method of obtaining anucleic acid sequence encoding a binding protein capable of binding atarget ligand, the method comprising the step of: (a) providing a Gramnegative bacterium comprising a nucleic acid sequence encoding acandidate binding protein, wherein the binding protein is expressed insoluble form in the bacterium; (b) contacting the bacterium with alabeled ligand capable of diffusing into the bacterium; (c) selectingthe bacterium based on the presence of the labeled ligand within thebacterium, wherein the ligand and the candidate binding protein arebound in the bacterium; and (d) cloning the nucleic acid sequenceencoding the candidate binding protein.

In another aspect of the invention, the binding protein expressed in thebacterium is further defined as expressed in soluble form in theperiplasm of the bacterium. The nucleic acid sequence encoding thebinding protein may still further be defined as encoding a nucleic acidsequence comprising the candidate binding protein sequence operablylinked to a leader sequence capable of directing expression of thecandidate binding protein in the periplasm. Potentially any Gramnegative bacterium could be used with the invention, including, forexample, an E. coli bacterium. In one embodiment of the invention, thenucleic acid sequence encoding a candidate binding protein may befurther defined as capable of being amplified following the selection.The invention may still further be defined as including removing labeledligand not bound to the candidate binding protein.

In yet another aspect, the invention comprises providing a population ofGram negative bacteria. In one embodiment of the invention, thepopulation of bacteria is further defined as collectively capable ofexpressing a plurality of candidate binding proteins. In yet anotherembodiment of the invention, the population of bacteria is obtained by amethod comprising the steps of: a) preparing a plurality DNA insertswhich collectively encode a plurality of different potential bindingproteins, and b) transforming a population of gram negative bacteriawith the DNA inserts. The population of Gram negative bacteria may bestill further defined as contacted with the labeled ligand.

In still yet another aspect of the invention, a candidate bindingprotein employed in accordance with the invention is further defined asan antibody or fragment thereof, or alternatively, is a binding proteinother than an antibody. Still further, the candidate binding protein maybe an enzyme, including any portion thereof. A candidate binding proteinused with the invention may be further defined as not capable ofdiffusing out of the periplasm in intact bacteria.

In still yet another aspect of the invention, a labeled ligand maycomprise a polypeptide, an enzyme and/or a nucleic acid or the like. Thelabeled ligand may be further defined as comprising a molecular weightof less than about 20,000 Da, less than about 10,000 Da or less thanabout 5,000 Da, and may in other embodiments of the invention bedescribed as greater than 600 Da in molecular weight. The labeled ligandmay be still further defined as fluorescently labeled.

In still yet another aspect, the invention comprises treating abacterium to facilitate diffusing of a target ligand into the periplasm.In certain embodiments of the invention, the treating may comprise useof hyperosmotic conditions, physical stress, treating the bacterium witha phage, or growing the bacterium at a sub-physiological temperature,for example, about 25° C.

In still yet another aspect, the invention comprises selecting one ormore bacteria using FACS or magnetic separation. In the invention, theligand and candidate binding protein may be further defined asreversibly bound in the periplasm.

In still yet another aspect, the invention provides a method ofobtaining a bacterium comprising a nucleic acid sequence encoding acatalytic protein catalyzing a chemical reaction involving a targetsubstrate, the method comprising the steps of: (a) providing a Gramnegative bacterium comprising a nucleic acid sequence encoding acandidate catalytic protein, wherein the catalytic protein is expressedin soluble form in the bacterium; (b) contacting the bacterium with atarget substrate capable of diffusing into the bacterium, wherein thecandidate catalytic protein catalyzes a chemical reaction involving thetarget substrate and wherein the chemical reaction yields at least afirst substrate product; and (c) selecting the bacterium based on thepresence of the first substrate product. In yet another aspect of theinvention, the method may be further defined as a method of obtaining anucleic acid sequence encoding a catalytic protein catalyzing a reactionwith a target substrate, the method further comprising the step of: (d)cloning the nucleic acid sequence encoding the candidate catalyticprotein. By “catalytic protein” it is meant a molecule which is capableof increasing the rate of a chemical reaction relative to the rate thereaction would occur absent the catalytic protein. In the method, thecandidate catalytic protein may be expressed in soluble form in theperiplasm of the bacterium. The nucleic acid sequence encoding acandidate catalytic protein may, in further embodiments of theinvention, be defined as operably linked to a leader sequence capable ofdirecting expression of the candidate catalytic protein in theperiplasm.

In still yet another aspect, the aforementioned method may be carriedout with any Gram negative bacterium, for example, an E. coli bacterium.The invention may also comprise providing a population of Gram negativebacteria. The population may be further defined as collectively capableof expressing a plurality of candidate catalytic proteins. In oneembodiment of the invention, the population of bacteria is obtained by amethod comprising the steps of: a) preparing a plurality DNA insertswhich collectively encode a plurality of different candidate catalyticproteins, and b) transforming a population of Gram negative bacteriawith the DNA inserts. The Gram negative bacteria may be defined ascontacted with the target substrate. A bacterium selected with theinvention may be further defined as viable following the selecting.Selecting may be carried out by any desired method, for example, FACS ormagnetic separation.

In still yet another aspect of the invention, a candidate catalyticprotein is an enzyme. The candidate catalytic protein may also bedefined as not capable of diffusing out of the periplasm.

In still yet another aspect of the invention, a target substrate maycomprise a molecule containing a scissile amide bond. The targetsubstrate may also comprise a polypeptide or a nucleic acid. In certainembodiments of the invention, the target substrate comprises a moleculecontaining a scissile carboxylic ester bond, a molecule containing ascissile phosphate ester bond, a molecule containing a scissilesulfonate ester bond, a molecule containing a scissile carbonate esterbond, a molecule containing a scissile carbamate bond, and/or a moleculecontaining a scissile thioester bond. In still further embodiments ofthe invention, the target substrate is further defined as comprising amolecular weight of less than about 20,000 Da, less than about 5,000 Da,less than about 3,000 Da, or may be defined as comprising a molecularweight of greater than about 600 Da, including from about 600 Da toabout 30,000 Da.

In still yet another aspect of the invention, the first substrateproduct is further defined as capable of being detected based on thepresence of a fluorescent signature. This fluorescent signature may beabsent in the target substrate, and may only be produced upon chemicalreaction to produce the first substrate product. In one embodiment ofthe invention, a fluorescent signature is produced by catalytic cleavageof a scissile bond. The method may be further defined as comprising useof a FRET system, the FRET system comprising a fluorophore bound by ascissile bond to at least a first molecule capable of quenching thefluorescence of the fluorophore, wherein cleavage of the scissile bondallows the first molecule to diffuse away from the fluorophore andwherein the fluorescence of the fluorophore becomes detectable. In themethod, the fluorophore may comprise a positive charge allowing thefluorophore to remain associated with the bacterium. In furtherembodiments of the invention, the target substrate may be furtherdefined as comprising a latent fluorescent moiety capable of beingreleased by the chemical reaction involving the target substrate. Thelatent fluorescent moiety released by the cleavage may possess anoverall positive charge allowing the moiety to remain associated withthe bacterium following the cleavage. In yet another embodiment of theinvention, the method may comprise labeling the target substrate with afluorescent pH probe capable of being detected upon a change in pHassociated with the chemical reaction involving the target substrate.The fluorescent pH probe may possess an overall positive charge allowingthe fluorescent pH probe to remain associated with the bacteriumfollowing the chemical reaction involving the target substrate. It willbe understood by those of skill in the art that multiple products may beproduced as a result of the chemical reaction and any one or more ofthese could potentially be detected to reveal that occurrence of thechemical reaction. Accordingly, the reaction product may comprise achange in pH within the bacterium which could be detected.

In still yet another aspect, the invention may comprise treating thebacterium to facilitate the diffusing into the periplasm, for example,using hyperosmotic conditions, physical stress, treating the bacteriumwith a phage, and growing the bacterium at a sub-physiologicaltemperature, for example, about 25° C.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. General scheme of the basis of a preferred embodiment of theinvention. A library of proteins expressed in the periplasmic space ofbacteria is contacted with a fluorescent reagent. Bacterial clonesexpressing a protein having a desired activity (e.g., either binding ofthe probe or enzymatic conversion to a product) become fluorescentlylabeled. The fluorescent cells can subsequently be isolated by FACS.

FIG. 2. Isolation of affinity improved mutants of an anti-digoxinantibody by two rounds of sorting. A library of scFv mutants in whichthree residues in the light chain had been randomized was constructed asdescribed in Example 2. A total of 2.5×10⁶ transformants were grown inliquid media, labeled with 100 nM digoxin-BODIPY™ and fluorescent cellsfalling within the window shown in the rightmost panel were sorted byFACS. The sorted cells were grown in liquid media, re-labeled and cellsfalling within the specified window as shown in the center panel wereisolated. Following a final round of re-growth the cells were analyzedby FACS (left-most panel). Single scFv antibody colonies were picked atrandom, analyzed and the affinity of the corresponding scFv proteins arereported in Table 1.

FIG. 3. Shows strain dependence of periplasmic FACS signal: i)TG1/pHEN2.thy; ii) HB2151/pHEN2.thy; iii) ABLE™C/pHEN2.thy; iv)ABLE™K/pHEN2.thy; v) TG1/pHEN2.dig; vi) HB2151/pHEN2.dig; vii)ABLE™C/pHEN2.dig; viii) ABLE™K/pHEN2.dig.

FIGS. 4A-4H. Effect of hyperosmotic shock on labeling efficiency: FIG.4A, FIG. 4C, FIG. 4E, FIG. 4G: pHEN2.thy; FIG. 4B, FIG. 4D, FIG. 4F,FIG. 4H pHEN2.dig; FIG. 4A and FIG. 4B, 1×PBS; FIG. 4C and FIG. 4D,2.5×PBS; FIG. 4E and FIG. 4F 5×PBS; FIG. 4G and FIG. 4H 10×PBS.

FIGS. 5A-5D. Maximizing periplasmic FACS signal in ABLE™C labeled in5×PBS using P_(tac) vector and superinfection with M13KO7 (moi of 10)0.5h pre-induction: FIG. 5A pHEN.thy; FIG. 5C pHEN2.thy/M13K07;pHEN2.dig; FIG. 5D pHEN2.dig/M13K07.

FIGS. 6A-6C. FIG. 6A: Phage eluate titers, after each round of panningFIG. 6B: Polyclonal phage ELISA of purified phage stocks ondigoxin-ovalbumin. FIG. 6C: FACScanning naïve library FIG. 6C-1 androunds one to five (FIG. 6C-2 to FIG. 6C-6) of panning on digoxin-BSAusing BODIPY™-digoxygenin.

FIG. 7A, B. Amino acid and nucleotide sequences of scFv antibodyfragments isolated by expression in the periplasm and FACS. FIG. 7A:Heavy chain of dig1 is shown in true font while dig3 is shown in italicsunderneath. The nucleotide sequences corresponding to the heavy chainsof dig 1 and dig 3 are given by SEQ ID NO:17 and SEQ ID NO:18,respectively. Dig2 variation from dig 1 is as indicated in underlinedtext within CDR3. FIG. 7B: Light chain of dig1, 2 and 3 with variationsin CDR3 indicated as for heavy chain. The nucleotide sequencescorresponding to the light chains of dig1 and dig 3 are given by SEQ IDNO:19 and SEQ ID NO:20, respectively. The underlined four nucleotidevariation beginning at nucleotide 99 is given by SEQ ID NO:21.

FIGS. 8A-8D. Labeling of periplasmic scFv by fluorescently taggedoligonucleotide probe. ABLE™C cells expressing periplasmic scFv specificfor either atrazine as a negative control (FIG. 8A and FIG. 8C) or fordigoxin (FIG. 8B and FIG. 8D) were labeled either with: 100 nM withdigoxigenin-BODIPY™(FIG. 8A and FIG. 8B) or 100nM of dig-5A-FL (FIG. 8Cand FIG. 8D). 10,000 events were recorded using a FACSort flow cytometerat a rate of approximately 1,000 events per second.

FIGS. 9A-9B. Fluorescence discrimination of E. coli expressing theenzyme cutinase (an esterase) from control bacteria not expressing theenzyme. E. coli DH5acells were transformed either with the plasmidpBAD18Cm (control cells) or with the derivative plasmid pKG3-53-1encoding the Fusarium solani enzyme cutinase. FIG. 9A. Fluorescencehistogram showing the selective labeling of E. coli expressing cutinasein the periplasm (pkg3-53-1 containing-cells) using a fluorescentesterase substrate (10μM Fluorescein Dibutyrate) for 30 minutes at 37°C. FIG. 9B. Fluorescence histogram of from selective labeling ofcutinase-expressing cells (transcribed from the pKG3-53-4 vector)labeled with a fluorescent pH-Sensitive Dye (1μM LysoSensor GreenDND-189) in the presence of cutinase substrate (1 mM 4-NitrophenylButyrate). The cells were labeled for 5 minutes at 25° C. Acidificationof the periplasm occurred as a result of ester hydrolysis by thecutinase.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present technology circumvents the limitations of the prior art andprovides an entirety novel means for the screening of very largepolypeptide libraries. In particular, the invention overcomesdeficiencies in the prior art by providing a rapid approach forisolating proteins that bind to small molecules and peptides via“display-less” library screening. A description of an example of such aprocess in accordance with the invention is described for illustrativepurposes in FIG. 1. In the technique, libraries of candidate bindingproteins, such as antibody sequences, are expressed in soluble form inthe periplasmic space of gram negative bacteria, such as Escherichiacoli, and are mixed with a labeled ligand. The periplasm comprises thespace defined by the inner and outer membranes of a gram-negativebacterium.

In wild-type E. coli and other gram negative bacteria, the outermembrane serves as a permeability barrier that severely restricts thediffusion of molecules greater than 600 Da into the periplasmic space(Decad and Nikado, 1976). Conditions that increase the permeability ofthe outer membrane, allowing larger molecules to diffuse in theperiplasm, have two deleterious effects in terms of the ability toscreen libraries: (a) the cell viability is affected to a significantdegree and (b) the diffusion of molecules into the cell is accompaniedby the diffusion of proteins and other macromolecules. The expressedproteins leak out of the periplasm and thus the resulting cell basedlibraries cannot be screened in a meaningful way. However, the inventorshave identified experimental conditions such that allow fluorescentconjugates of ligands and polypeptides equilibrate across the outermembrane while proteins secreted into the periplasm remain within thecell. Therefore, in bacterial cells expressing recombinant polypeptideswith affinity for the ligand, the concentration of the labeled ligandbound to the binding protein is increased, allowing the bacteria to beisolated from the rest of the library. Where fluorescent labeling of thetarget ligand is used, cells may efficiently be isolated by fluorescenceactivated cell sorting (FACS). Thus, in effect, the cell envelope servesas a “dialysis bag” that retains macromolecule:ligand complexes, but notfree ligand (FIG. 1). With this approach, existing libraries of secretedproteins in bacteria can be easily tested for ligand binding without theneed for subcloning into a phage or cell surface display system.

I. Anchor-Less Display Library Screening

Prior art methods of both phage display and bacterial cell surfacedisplay suffer from a limitation in that the protein is required, bydefinition, to be physically displayed on the surface of the vehicleused, to allow unlimited access to the targets (immobilized for phage orfluorescently conjugated ligands for FACS) (U.S. Pat. No. 5,223,409, thedisclosure of which is specifically incorporated herein by reference inits entirety). Certain proteins are known to be poorly displayed onphage (Maenaka et al., 1996; Corey et al., 1993) and the toxic effectsof cell surface display have been treated at length (Daugherty et al.,1999). The proteins to be displayed also need to be expressed as fusionproteins, which may alter their function. The selection constraintsimposed by any display system may, therefore, limit the application torelatively small and “simple” proteins and deny access to a multitude oflarge and complex multisubunit species. The latter are very likely to beincapable of partaking efficiently in the complex process of phageassembly termination or outer-membrane translocation without veryserious effects on host cell viability.

Herein, the inventors have described conditions whereby expressedbinding proteins, for example, an antibody, are targeted to theperiplasmic compartment of E. coli and yet are amenable to bindingligands and peptides of up to at least 2 kDa. As used herein, the term“binding protein” includes not only antibodies, but also fragments ofantibodies, as well as any other polypeptide or protein potentiallycapable of binding a given target molecule. The antibody or otherbinding proteins may be expressed with the invention directly and not asfusion proteins. Such a technique may be termed “anchor-less-display”(ALD). To understand how it may work, one needs to be aware of thelocation in which it functions.

The periplasmic compartment is contained between the inner and outermembranes of Gram negative cells (see, e.g., Oliver, 1996). As asub-cellular compartment, it is subject to variations in size, shape andcontent that accompany the growth and division of the cell. Within aframework of peptidoglycan heteroploymer is a dense mileau ofperiplasmic proteins and little water, lending a gel-like consistency tothe compartment (Hobot et al., 1984; van Wielink and Duine, 1990). Thepeptidoglycan is polymerized to different extents depending on theproximity to the outer membrane, close-up it forms the murein sacculusthat affords cell shape and resistance to osmotic lysis.

The outer membrane (see Nikaido, 1996) is composed of phospholipids,porin proteins and, extending into the medium, lipopolysaccharide (LPS).The molecular basis of outer membrane integrity resides with LPS abilityto bind divalent cations (Mg2+ and Ca2+) and link each otherelectrostatically to form a highly ordered quasi-crystalline ordered“tiled roof” on the surface (Labischinski et al., 1985). The membraneforms a very strict permeability barrier of allowing passage ofmolecules no greater than around 650 Da (Burman et al., 1972; Decad andNikaido, 1976) via the porins. The large water filled porin channels areprimarily responsible for allowing free passage of mono anddisaccharides, ions and amino acids in to the periplasm compartment(Naeke, 1976; Nikaido and Nakae, 1979; Nikaido and Vaara, 1985). Withsuch strict physiological regulation of access by molecules to theperiplasm it may appear, at first glance, inconceivable that ALD shouldwork unless the ligands employed are at or below the 650 Da exclusionlimit or are analogues of normally permeant compounds. However, theinventors have shown that ligands at least 2000 Da in size can diffuseinto the periplasm. Such diffusion can be aided by one or moretreatments of a bacterial cell, thereby rendering the outer membranemore permeable, as is described herein below.

II. Ligand Access to the Bacterial Periplasm

Certain classes of hydrophobic antibiotics, larger than the 650 Daexclusion limit, can diffuse through the bacterial outer membraneitself, independent of membrane porins (Farmer et al., 1999). Theprocess may actually permeabilize the membrane on so doing (Jouenne andJunter, 1990). Such a mechanism has been adopted to selectively labelthe periplasmic loops of a cytoplasmic membrane protein in vivo with apolymyxin B nonapeptide (Wada et al., 1999). Also, certain long chainphosphate polymers (100 Pi) appear to bypass the normal molecularsieving activity of the outer membrane altogether (Rao and Torriani,1988). However, such conditions generally lead to a decrease in cellviability. Maintaining the cells in a viable state is essential forlibrary screening applications since non-viable cells cannot bepropagated.

The inventors have defined conditions that lead to the permeation ofligands into the periplasm without loss of viability or release of theexpressed proteins from the cells. As a result, cells expressing bindingprotein can be fluorescently labeled simply by incubating with asolution of fluorescently labeled ligand. The inventors have observedmarked differences in labeling efficiencies of different strains ofbacterial host cells. It has been shown previously that increasedpermeability due to OmpF overexpression was caused by the absence of ahistone like protein resulting in a decrease in the amount of a negativeregulatory MRNA for OmpF translation (Painbeni et al., 1997). Also, DNAreplication and chromosomal segregation is known to rely on intimatecontact of the replisome with the inner membrane, which itself contactsthe outer membrane at numerous points. That the FACS optimal ABLECstrain has mutations altering plasmid copy number is thus, noteworthy.

The inventors also noticed that treatments such as hyperosmotic shockcan improve labeling significantly. It is known that many agentsincluding, calcium ions (Bukau et al., 1985) and even Tris buffer (Irvinet al., 1981) alter the permeability of the outer-membrane. Further, theinventors found that phage infection stimulates the labeling process.Both the filamentous phage inner membrane protein pIII and the largemultimeric outer membrane protein pIV can alter membrane permeability(Boeke et al., 1982) with mutants in pIV known to improve access tomaltodextrins normally excluded (Marciano et al., 1999). Using thetechniques of the invention comprising a judicious combination ofstrain, salt and phage, the inventors surpassed the highest fluorescentsignal reported even for cell surface display of the antidigoxinantibody scFv (Daugherty et al., 1999). This result demonstrated thatthe anchor-less display methodology allows for excellentligand-dependent labeling of cells. Cells labeled with a fluorescentligand can then be easily isolated from cells that express non-bindingprotein mutants using flow cytometry or other related techniques.

III. Periplasmic Peptide Expression

In one embodiment of the invention, bacterial cells are providedexpressing candidate molecules in the periplasm of the bacteria. Anadvantage of the instant invention is that, unlike prior art phagedisplay techniques, it is not necessary that the candidate molecule besurface-bound, thereby limiting the potential for effects due to surfaceinteractions with the candidate molecule, or limitations in theexpression thereof. Thus, the invention employs “anchor-less display.”The general scheme behind the technique of the invention is theadvantageous expression of a heterogeneous collection of peptides insoluble form in the periplasm.

Methods that may be employed with the current invention for theexpression of heterologous proteins in the periplasm of Gram negativebacteria are well known in the art (see, for example, U.S. Pat. Nos.5,646,015 and 5,759,810, the disclosures of which are incorporatedherein by reference in their entirety). In such techniques,bacterial-encoded heterologous proteins can be directed across the innermembrane of the bacterial cell envelope, into the space between theinner and the outer membrane known as the periplasm. For example, when aprotein is expressed as a fusion protein having an E. coli-recognizedpeptide or “signal peptide” attached to its N-terminus, the desiredprotein is secreted into the periplasm. Signal peptides that couldpotentially be employed with the invention are well known in the art andinclude, for example, those described by Watson (1984), Oka et al,(1985), Hsiung et al, (1986) and EP 177,343, each of the disclosures ofwhich are specifically incorporated herein by reference in theirentirety.

In phage display, a gene encoding a protein of interest is commonlylinked to the amino-terminal domain of the gene III coat protein of thefilamentous phage M13, or another surface-associated molecule. Thefusion is mutated to form a library of structurally related fusionproteins that are expressed in low quantity on the surface of phagemidcandidates. For example, U.S. Pat. No. 5,571,698 describes directedevolution using an M13 phagemid system. However, in the instantinvention, fusion to the gene III coat protein is not necessary, as theprotein is expressed in soluble form in the periplasm. Instead, it maybe desirable to create a fusion protein of the candidateperiplasmic-expressed binding protein or antibody with a signal sequencedirecting expression of that protein in the periplasm. As such,techniques for the creation of heterogeneous collections of candidatemolecules, well known to those of skill in the art in conjunction withphage display, could be adapted for use with the invention. Those ofskill in the art will recognize that such adaptations will include theuse of bacterial elements for expression and secretion of candidatemolecules into the periplasm, including, promoter, enhancers or leadersequences. The current invention provides the advantage relative tophage display of not requiring the creation of fusions withsurface-associated molecules, as required in standard display protocols,which may be poorly expressed or may be deleterious to the host cell.

Examples of techniques that could be employed in conjunction with theinvention for expression of candidate binding proteins and/orantibodies, in the periplasm include the techniques for expression ofimmunoglobulin heavy chain libraries described in U.S. Pat. No.5,824,520. In this technique, a single chain antibody library isgenerated by creating highly divergent, synthetic hypervariable regions.Similar techniques for antibody display are given by U.S. Pat. No.5,922,545.

In accordance with another embodiment of the invention, theidentification and selection of novel substrates for enzymes also couldbe carried out in the bacterial periplasm (see, for example, U.S. Pat.No. 5,780,279). The method comprises constructing a gene fusioncomprising DNA encoding a polypeptide fused to a DNA encoding asubstrate peptide. The DNA encoding the substrate peptide is mutated atone or more codons, thereby generating a family of mutants. The fusionprotein could be expressed in the periplasm of a bacterium and subjectedto potential inhibition or modification by target ligands. Thosebacteria in which modifications have taken place can then be separatedfrom those that have not. By employing FACS screening technology, thegeneral progress of a reaction could similarly be efficiently monitored.

IV. Screening Candidate Molecules

The present invention further comprises methods for identifyingmolecules capable of binding a target ligand. The molecules screened maycomprise large libraries of diverse candidate substances, or,alternatively, may comprise particular classes of compounds selectedwith an eye towards structural attributes that are believed to make themmore likely to bind the target ligand. In a preferred embodiment of theinvention, the candidate molecule is an antibody, or a fragment orportion thereof In other embodiments of the invention, the candidatemolecule may be another binding protein or an enzyme.

To identify a candidate molecule capable of binding a target ligand inaccordance with the invention, one may carry out the steps of: providinga population of Gram negative bacterial cells comprising candidatemolecules expressed in the periplasm of the bacteria; admixing thebacteria and at least a first labeled target ligand capable of diffusinginto the periplasm of the bacteria; and identifying at least a firstbacterium expressing a molecule capable of binding the target ligand.

In the aforementioned method, the binding between the candidate moleculeand the ligand will prevent the diffusing out of the cell. In this way,multiple molecules of the labeled ligand will be retained in theperiplasm of the bacterium. The labeling may then be used to isolate thecell expressing the molecule capable of binding the target ligand, andin this way, the gene encoding the molecule isolated. The moleculecapable of binding the target ligand may then be produced in largequantities using in vivo or ex vivo expression methods, and then usedfor any desired application, for example, for diagnostic or therapeuticapplications, as described below.

As used herein the term “candidate molecule” or “candidate polypeptide”refers to any molecule or polypeptide that may potentially have affinityfor a target ligand. The candidate substance may be a protein orfragment thereof, including a small molecule. The candidate molecule mayin one embodiment of the invention, comprise an antibody sequence orfragment thereof Such sequences may be particularly designed for thelikelihood that they will bind a target ligand.

Binding proteins or antibodies isolated in accordance with the inventionalso may help ascertain the structure of a target ligand. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies to a functional, pharmacologicallyactive antibody. As a mirror image of a mirror image, the binding siteof anti-idiotype would be expected to be an analog of the originalantigen. The anti-idiotype could then be used to identify and isolatepeptides from banks of chemically- or biologically-produced peptides.Selected peptides would then serve as the pharmacore. Anti-idiotypes maybe generated using the methods described herein for producingantibodies, using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for binding the target ligand. Such libraries could be providedby way of nucleic acids encoding the small molecules or bacteriaexpressing the molecules.

V. Screening of Enzyme Libraries

Yet another aspect of the present invention relates to the isolation ofenzyme catalysts capable of catalyzing the conversion of a reactionsubstrate to a product. The inventors have discovered that a number offluorescent substrates for enzymatic reactions can permeate into theperiplasmic space of E. coli. Cells that express an enzyme capable ofreacting with such a substrates produce fluorescent products in directproportion to the catalytic activity of the protein. The inventors havefurther found that the fluorescent product of the enzymatic reaction isretained within the cell. As a result, cells become fluorescentlystained in proportion to the catalytic activity of the expressed enzymeand can be sorted from a population of mutant proteins by FACS.

Flow cytometry is well suited for the analysis of enzyme activity andkinetics at the single cell level. The inventors have discovered thatmany fluorescent substrates that normally cannot permeate into thecytoplasm can nonetheless freely diffuse into the periplasm of bacterialcells. Cells expressing an enzyme in the bacterial periplasm are thuscapable of converting the substrate into the respective fluorescentproduct. The inventors further discovered, unexpectedly, that followingthe enzymatic reaction the fluorescent product is selectively retainedwithin the periplasm and thus the cell becomes fluorescent and can beisolated by FACS.

In the case of the LysoSensor Green DND-189 in the presence of 1 mM4-nitrophenyl butyrate reaction, it is not a fluorescent product that isbeing detected. Rather, the LysoSensor Green DND-189 is a pH-sensitivedye, and as the enzyme catalyzes the conversion of 4-nitrophenylbutyrate to 4-nitrophenol and butyric acid in the periplasm, the pHdrops in unbuffered solution. This drop in pH is being detected asincreased fluorescence by the LysoSensor Green DND-189, which remainsassociated with the cell via electrostatic attraction. Here, theLysoSensor Green DND-189 becomes protonated at lower pH and thuspositively-charged, so it associates with the negatively-chargedbacterial outer membrane long enough to allow FACS isolation.

1. Cloning of Binding Protein Coding Sequences

The binding affinity of an antibody or another binding protein can, forexample, be determined by the Scatchard analysis of Munson & Pollard(1980). After a bacterial cell is identified that produces molecules ofthe desired specificity, affinity, and/or activity, the correspondingcoding sequence may be cloned. In this manner, DNA encoding the moleculecan be isolated and sequenced using conventional procedures (e.g. byusing oligonucleotide probes that are capable of binding specifically togenes encoding the antibody or binding protein).

Once isolated, the antibody or binding protein DNA may be placed intoexpression vectors, which can then transfected into host cells such assimian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of binding protein in the recombinant host cells. The DNA alsomay be modified, for example, by substituting the coding sequence forhuman heavy and light chain constant domains in place of the homologousmurine sequences (Morrison, et al., 1984), or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. In that manner, “chimeric” or“hybrid” binding proteins are prepared that have the desired bindingspecificity.

Typically, such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity the target ligand and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

2. Maximization of Protein Affinity for Ligands

In a natural immune response, antibody genes accumulate mutations at ahigh rate (somatic hypermutation). Some of the changes introduced willconfer higher affinity, and B cells displaying high-affinity surfaceimmunoglobulin. This natural process can be mimicked by employing thetechnique known as “chain shuffling” (Marks et al., 1992). In thismethod, the affinity of “primary” human antibodies obtained inaccordance with the invention could be improved by sequentiallyreplacing the heavy and light chain V region genes with repertoires ofnaturally occurring variants (repertoires) of V domain genes obtainedfrom unimmunized donors. This technique allows the production ofantibodies and antibody fragments with affinities in the nM range. Astrategy for making very large antibody repertoires was described byWaterhouse et al., (1993), and the isolation of a high affinity humanantibody directly from such large phage library was reported by Griffithet al., (1994). Gene shuffling also can be used to derive humanantibodies from rodent antibodies, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting”, theheavy or light chain V domain gene of rodent antibodies obtained by thephage display technique is replaced with a repertoire of human V domaingenes, creating rodent-human chimeras. Selection on the antigen resultsin isolation of human variable regions capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

3. Labeled Ligands

In one embodiment of the invention, an antibody or binding protein isisolated which has affinity for a labeled ligand. Such a labeled ligandis, in one embodiment of the invention, preferably less that 50,000 Dain size in order to allow efficient diffusion of the ligand into thebacterial periplasm. As indicated above, it will typically be desired inaccordance with the invention to provide a ligand which has been labeledwith one or more detectable agent(s). This can be carried out, forexample, by linking the ligand to at least one detectable agent to forma conjugate. For example, it is conventional to link or covalently bindor complex at least one detectable molecule or moiety. A “label” or“detectable label” is a compound and/or element that can be detected dueto specific functional properties, and/or chemical characteristics, theuse of which allows the ligand to which it is attached to be detected,and/or further quantified if desired. Examples of labels which could beused with the invention include, but are not limited to, enzymes,radiolabels, haptens, fluorescent labels, phosphorescent molecules,chemiluminescent molecules, chromophores, luminescent molecules,photoaffinity molecules, colored particles or ligands, such as biotin.

In a preferred embodiment of the invention, a visually-detectable markeris used such that automated screening of cells for the label can becarried out. In particular, fluorescent labels are preferred in thatthey allow use of FACS for isolation of cells expressing a desiredbinding protein or antibody. Examples of agents that may be detected byvisualization with an appropriate instrument are known in the art, asare methods for their attachment to a desired ligand (see, e.g., U.S.Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated hereinby reference). Such agents can include paramagnetic ions; radioactiveisotopes; fluorochromes; NMR-detectable substances and substances forX-ray imaging. Types of fluorescent labels that may be used with theinvention will be well known to those of skill in the art and include,for example, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665,BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3,Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488,Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green,Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine,and/or Texas Red.

Examples of paramagnetic ions that could be used as labels include ionssuch as chromium (III), manganese (II), iron (III), iron (II), cobalt(II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III). Ions useful inother contexts include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

Another type of ligand conjugates contemplated in the present inventionare those where the ligand is linked to a secondary binding moleculeand/or to an enzyme (an enzyme tag) that will generate a colored productupon contact with a chromogenic substrate. Examples of such enzymesinclude urease, alkaline phosphatase, (horseradish) hydrogen peroxidaseor glucose oxidase. In such instances, it will be desired that cellsselected remain viable. Preferred secondary binding ligands are biotinand/or avidin and streptavidin compounds. The use of such labels is wellknown to those of skill in the art and are described, for example, inU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241; each incorporated herein by reference.

Molecules containing azido groups also may be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide-bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide-binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used as ligandbinding agents.

Labeling can be carried out by any of the techniques well known to thoseof skill in the art. For instance, ligands can be labeled by contactingthe ligand with the desired label and a chemical oxidizing agent such assodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Similarly, a ligand exchange process could be used.Alternatively, direct labeling techniques may be used, e.g., byincubating the label, a reducing agent such as SNCl₂, a buffer solutionsuch as sodium-potassium phthalate solution, and the ligand.Intermediary functional groups on the ligand could also be used, forexample, to bind labels to a ligand in the presence ofdiethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Other methods are also known in the art for the attachment orconjugation of a ligand to its conjugate moiety. Some attachment methodsinvolve the use of an organic chelating agent such adiethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3α-6α-diphenylglycouril-3 attached to the ligand (U.S. Pat.Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).Ligands also may be reacted with an enzyme in the presence of a couplingagent such as glutaraldehyde or periodate. Conjugates with fluoresceinmarkers can be prepared in the presence of these coupling agents or byreaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging ofbreast tumors is achieved using monoclonal antibodies and the detectableimaging moieties are bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

V. Illustrative Use of the Invention

The examples described herein demonstrate the use of the invention forthe affinity maturation of the anti-cardiac glycoside scFv 26.10, andthe de novo isolation of antibodies from large repertoire libraries.Importantly the current invention results in the isolation of proteinvariants that are missed by other protein library screening technologiessuch as phage display. As discussed above, most scFv are toxic to thehost cell when targeted to the periplasm. Although reasonable expressionlevels of such molecules may be obtained by altering culture conditions,provision of chaperones and foldases etc., the host cells are often notrecoverable after expression. FACS selection with theanchor-less-display (ALD) techniques of the invention requires a highenough expression level for a strong FACS signal but not so high as toirreversibly damage the cell and prevent its isolation. In contrast,phage display imposes little selection for expression. As a result,proteins isolated from a phage display library screening program areoften very difficult to produce in sufficient quantities for furthercharacterization. The advent of highly expressable framework-basedlibraries (Knappick et al., 2000) should help circumvent the expressionversus viability problem. The high sort-rates of current FACS machinesgenerally enable the screening of very large libraries (10⁹-10¹⁰) andcan completely circumvent phage or cell surface display.

The ability to specifically label periplasmic expressed proteins withappropriate fluorescent ligands also has applications other than libraryscreening. Specifically labeling with fluorescent ligands and flowcytometry can be used for monitoring production during proteinmanufacturing. While flow cytometry has been used previously for theanalysis of bacterial cells, it has not been used for the specificlabeling and quantitation of periplasmic proteins. However, a largenumber of commercially important proteins including IGF-1 severalinterleukins, enzymes such as urokinase-type plasminogen activator,antibody fragments, inhibitors (e.g., Bovine pancreatic trypsininhibitor) are expressed in recombinant bacteria in a form secreted intothe periplasmic space. The level of production of such proteins withineach cell in a culture can be monitored by utilizing an appropriatefluorescent ligand and flow cytometric analysis, according to thetechniques taught by the present invention.

Generally, monitoring protein expression requires cell lysis anddetection of the protein by immunological techniques or followingchromatographic separation. However, ELISA or western blot analysis istime-consuming and does not provide information on the distribution ofexpression among a cell population and cannot be used for on-linemonitoring (Thorstenson et al., 1997; Berrier et al., 2000). Incontrast, FACS labeling is rapid and simple and can well be applied toonline monitoring of industrial size fermentations of recombinantproteins expressed in gram-negative bacteria. Similarly, the inventioncould be used to monitor the production of a particular byproduct of abiological reaction. This also could be used to measure the relativeconcentration or specific activity of an enzyme expressed in vivo in abacterium or provided ex vivo. The passive and instant nature of ALDprovides the advantage of allowing instant analysis of a population ofcells with direct observations rather than relying on extensive indirectprotocols.

Once a ligand-binding protein, such as an antibody, has been isolated inaccordance with the invention, it may be desired to link the molecule toat least one agent to form a conjugate to enhance the utility of thatmolecule. For example, in order to increase the efficacy of antibodymolecules as diagnostic or therapeutic agents, it is conventional tolink or covalently bind or complex at least one desired molecule ormoiety. Such a molecule or moiety may be, but is not limited to, atleast one effector or reporter molecule. Effector molecules comprisemolecules having a desired activity, e.g., cytotoxic activity.Non-limiting examples of effector molecules which have been attached toantibodies include toxins, anti-tumor agents, therapeutic enzymes,radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety which may be detected usingan assay. Techniques for labeling such a molecule are known to those ofskill in the art and have been described herein above.

Labeled binding proteins such as antibodies which have been prepared inaccordance with the invention may also then be employed, for example, inimmunodetection methods for binding, purifying, removing, quantifyingand/or otherwise generally detecting biological components such asprotein(s), polypeptide(s) or peptide(s). Some immunodetection methodsinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescentassay, bioluminescent assay, and Western blot to mention a few. Thesteps of various useful immunodetection methods have been described inthe scientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,1999; Gulbis B and Galand P, 1993; and De Jager R et al., 1993, eachincorporated herein by reference. Such techniques include binding assayssuch as the various types of enzyme linked immunosorbent assays (ELISAs)and/or radioimmunoassays (RIA) known in the art.

The ligand-binding molecules, including antibodies, prepared inaccordance with the present invention may also, for example, inconjunction with both fresh-frozen and/or formalin-fixed,paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, and/or is well known to those ofskill in the art (Abbondanzo et al, 1990).

VI. Automated Screening with FACS

In one embodiment of the invention, fluorescence activated cell sorting(FACS) screening or other automated flow cytometric techniques may beused for the efficient isolation of a bacterial cell comprising alabeled ligand bound to a candidate molecule in the periplasm of thebacteria. Instruments for carrying out FACS are known to those of skillin the art and are commercially available to the public. Examples ofsuch instruments include FACS Star Plus, FACScan and FACSort instrumentsfrom Becton Dickinson (Foster City, Calif.) Epics C from Coulter EpicsDivision (Hialeah, Fla.) and MoFlo from Cytomation (Colorado Springs,Colo.).

Flow cytometric techniques in general involve the separation of cells orother particles in a liquid sample. Typically, the purpose of flowcytometry is to analyze the separated particles for one or morecharacteristics thereof, for example, presence of a labeled ligand orother molecule. The basis steps of flow cytometry involve the directionof a fluid sample through an apparatus such that a liquid stream passesthrough a sensing region. The particles should pass one at a time by thesensor and are categorized base on size, refraction, light scattering,opacity, roughness, shape, fluorescence, etc.

Rapid quantitative analysis of cells proves useful in biomedicalresearch and medicine. Apparati permit quantitative multiparameteranalysis of cellular properties at rates of several thousand cells persecond. These instruments provide the ability to differentiate amongcell types. Data are often displayed in one-dimensional (histogram) ortwo-dimensional (contour plot, scatter plot) frequency distributions ofmeasured variables. The partitioning of multiparameter data filesinvolves consecutive use of the interactive one- or two-dimensionalgraphics programs.

Quantitative analysis of multiparameter flow cytometric data for rapidcell detection consists of two stages: cell class characterization andsample processing. In general, the process of cell classcharacterization partitions the cell feature into cells of interest andnot of interest. Then, in sample processing, each cell is classified inone of the two categories according to the region in which it falls.Analysis of the class of cells is very important, as high detectionperformance may be expected only if an appropriate characteristic of thecells is obtained.

Not only is cell analysis performed by flow cytometry, but so too issorting of cells. In U.S. Pat. No. 3,826,364, an apparatus is disclosedwhich physically separates particles, such as functionally differentcell types. In this machine, a laser provides illumination which isfocused on the stream of particles by a suitable lens or lens system sothat there is highly localized scatter from the particles therein. Inaddition, high intensity source illumination is directed onto the streamof particles for the excitation of fluorescent particles in the stream.Certain particles in the stream may be selectively charged and thenseparated by deflecting them into designated receptacles. A classic formof this separation is via fluorescent-tagged antibodies, which are usedto mark one or more cell types for separation.

Other methods for flow cytometry can be found in U.S. Pat. Nos.4,284,412; 4,989,977; 4,498,766; 5,478,722; 4,857,451; 4,774,189;4,767,206; 4,714,682; 5,160,974; and 4,661,913, each of the disclosuresof which are specifically incorporated herein by reference.

VII. Nucleic Acid-Based Expression Systems

Nucleic acid-based expression systems may find use, in certainembodiments of the invention, for the expression of recombinantproteins. For example, one embodiment of the invention involvestransformation of Gram negative bacteria with the coding sequences ofcandidate antibody or other binding proteins having affinity for aselected ligand and the expression of such candidate molecules in theperiplasm of the Gram negative bacteria. In other embodiments of theinvention, expression of such coding sequences may be carried, forexample, in eukaryotic host cells for the preparation of isolatedbinding proteins having specificity for the target ligand. The isolatedprotein could then be used in one or more therapeutic or diagnosticapplications.

1. Methods of Nucleic Acid Delivery

Certain aspects of the invention may comprise delivery of nucleic acidsto target cells. For example, bacterial host cells may be transformedwith nucleic acids encoding candidate molecules potentially capablebinding a target ligand, In particular embodiments of the invention, itmay be desired to target the expression to the periplasm of thebacteria. Transformation of eukaryotic host cells may similarly find usein the expression of various candidate molecules identified as capableof binding a target ligand.

Suitable methods for nucleic acid delivery for transformation of a cellare believed to include virtually any method by which a nucleic acid(e.g., DNA) can be introduced into such a cell, or even an organellethereof Such methods include, but are not limited to, direct delivery ofDNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and5,580,859, each incorporated herein by reference), includingmicroinjection (Harlan and Weintraub, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference); by calcium phosphateprecipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;Rippe et al., 1990); by using DEAE-dextran followed by polyethyleneglycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987);by liposome mediated transfection (Nicolau and Sene, 1982; Fraley etal., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989;Kato et al, 1991); by microprojectile bombardment (PCT Application Nos.WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055,5,550,318, 5,538,877 and 5,538,880, and each incorporated herein byreference); by agitation with silicon carbide fibers (Kaeppleret al.,1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated hereinby reference); by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,591,616 and 5,563,055, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

a. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into a cell via electroporation. Electroporation involves theexposure of a suspension of cells and DNA to a high-voltage electricdischarge. In some variants of this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells (U.S. Pat. No. 5,384,253,incorporated herein by reference). Alternatively, recipient cells can bemade more susceptible to transformation by mechanical wounding.

b. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

2. Vectors

Vectors may find use with the current invention, for example, in thetransformation of a gram negative bacterium with a nucleic acid sequenceencoding a candidate polypeptide which one wishes to screen for abilityto bind a target ligand. In one embodiment of the invention, an entireheterogeneous “library” of nucleic acid sequences encoding targetpolypeptides may be introduced into a population of bacteria, therebyallowing screening of the entire library. The term “vector” is used torefer to a carrier nucleic acid molecule into which a nucleic acidsequence can be inserted for introduction into a cell where it can bereplicated. A nucleic acid sequence can be “exogenous,” which means thatit is foreign to the cell into which the vector is being introduced orthat the sequence is homologous to a sequence in the cell but in aposition within the host cell nucleic acid in which the sequence isordinarily not found. Vectors include plasmids, cosmids, viruses(bacteriophage, animal viruses, and plant viruses), and artificialchromosomes (e.g., YACs). One of skill in the art may construct a vectorthrough standard recombinant techniques, which are described in Maniatiset al., 1988 and Ausubel et al., 1994, both of which references areincorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

a. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthat the control sequences that direct transcription and/or expressionof sequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. One example of suchpromoter that may be used with the invention is the E. coli arabinosepromoter. Those of skill in the art of molecular biology generally arefamiliar with the use of promoters, enhancers, and cell typecombinations for protein expression, for example, see Sambrook et al.(1989), incorporated herein by reference. The promoters employed may beconstitutive, tissue-specific, inducible, and/or usefull under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

b. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

C. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see Carbonelli et al, 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

d. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments, a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, rhpdependent or rho independent terminators. In certain embodiments, thetermination signal may be a lack of transcribable or translatablesequence, such as due to a sequence truncation.

e. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

f. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

3. Host Cells As used herein, the terms “cell,” “cell line,” and “cellculture” may be used interchangeably. All of these terms also includetheir progeny, which is any and all subsequent generations. It isunderstood that all progeny may not be identical due to deliberate orinadvertent mutations. In the context of expressing a heterologousnucleic acid sequence, “host cell” refers to a prokaryotic or eukaryoticcell, and it includes any transformable organism that is capable ofreplicating a vector and/or expressing a heterologous gene encoded by avector. A host cell can, and has been, used as a recipient for vectors.A host cell may be “transfected” or “transformed,” which refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A transformed cell includes the primary subject celland its progeny.

In particular embodiments of the invention, a host cell is a gramnegative bacterial cell. These bacteria are suited for use with theinvention in that they posses a periplasmic space between the inner andouter membrane. As such, any other cell with such a periplasmic spacecould be used in accordance with the invention. Examples of Gramnegative bacteria that may find use with the invention may include, butare not limited to, E. coli, Pseudomonas aeruginosa, Vibrio cholera,Salmonella typhimurium, Shigellaflexneri, Haemophilus influenza,Bordotella pertussi, Erwinia amylovora, Rhizobium sp. The Gram negativebacterial cell may be still further defined as bacterial cell which hasbeen transformed with the coding sequence of a candidate polypeptidecapable of binding a selected ligand. The polypeptide will be expressedin the periplasmic space, and may comprise an antibody coding sequenceor another sequence. One means for expression of the polypeptide in theperiplasm is by attaching a leader sequence to the polypeptide capableof causing such directing.

Numerous prokaryotic cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (available on the world wideweb at atcc.org). An appropriate host can be determined by one of skillin the art based on the vector backbone and the desired result. Aplasmid or cosmid, for example, can be introduced into a prokaryote hostcell for replication of many vectors. Bacterial cells used as host cellsfor vector replication and/or expression include DH5.alpha., JM109, andKC8, as well as a number of commercially available bacterial hosts suchas SURE.RTM. Competent Cells and SOLOPACK.TM. Gold Cells(STRATAGENE.RTM., La Jolla). Alternatively, bacterial cells such as E.coli LE392 could be used as host cells for bacteriophage.

Many host cells from various cell types and organisms are available andwould be known to one of skill in the art. Similarly, a viral vector maybe used in conjunction with either a eukaryotic or prokaryotic hostcell, particularly one that is permissive for replication or expressionof the vector. Some vectors may employ control sequences that allow itto be replicated and/or expressed in both prokaryotic and eukaryoticcells. One of skill in the art would further understand the conditionsunder which to incubate all of the above described host cells tomaintain them and to permit replication of a vector. Also understood andknown are techniques and conditions that would allow large-scaleproduction of vectors, as well as production of the nucleic acidsencoded by vectors and their cognate polypeptides, proteins, orpeptides.

4. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Such systems could be used, forexample, for the production of a polypeptide product identified inaccordance with the invention as capable of binding a particular ligand.Prokaryote- and/or eukaryote-based systems can be employed for use withthe present invention to produce nucleic acid sequences, or theircognate polypeptides, proteins and peptides. Many such systems arecommercially and widely available.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INITROGEN® which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

5. Candidate Binding Proteins and Antibodies

In certain aspects of the invention, candidate antibodies or otherrecombinant proteins potentially capable of binding a target ligand areexpressed in the periplasm of a host bacterial cell. By expression of aheterogeneous population of such antibodies, those antibodies having ahigh affinity for a target ligand may be identified. The identifiedantibodies may then be used in various diagnostic or therapeuticapplications, as described herein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. The term“antibody” is also used to refer to any antibody-like molecule that hasan antigen binding region, and includes antibody fragments such as Fab′,Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (single chainFv), and engineering multivalent antibody fragments such as dibodies,tribodies and multibodies. The techniques for preparing and usingvarious antibody-based constructs and fragments are well known in theart. Means for preparing and characterizing antibodies are also wellknown in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

Once an antibody having affinity for a target ligand is identified, theantibody may be purified, if desired, using filtration, centrifugationand various chromatographic methods such as HPLC or affinitychromatography. Fragments of such antibodies can be obtained from theantibodies so produced by methods which include digestion with enzymes,such as pepsin or papain, and/or by cleavage of disulfide bonds bychemical reduction. Alternatively, antibody fragments encompassed by thepresent invention can be synthesized using an automated peptidesynthesizer.

A molecular cloning approach comprises one suitable method for thegeneration of a heterogeneous population of candidate antibodies thatmay then be screened in accordance with the invention for affinity totarget ligands. In one embodiment of the invention, combinatorialimmunoglobulin phagemid can be prepared from RNA isolated from thespleen of an animal. By immunizing an animal with the ligand to bescreened, the assay may be targeted to the particular antigen. Theadvantages of this approach over conventional techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination which further increases the chance of findingappropriate antibodies.

VIII. Manipulation and Detection of Nucleic Acids

In certain embodiments of the invention it may be desired to employ oneor more techniques for the manipulation and/or detection of nucleicacids. Such techniques may include, for example, the preparation ofvectors for transformation of host cells as well as methods for cloningselected nucleic acid segments from a transgenic cells. Methodology forcarrying out such manipulations will be well known to those of skill inthe art in light of the instant disclosure.

1. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis may beperformed on whole cell or tissue homogenates or biological fluidsamples without substantial purification of the template nucleic acid.The nucleic acid may be genomic DNA or fractionated or whole cell RNA.Where RNA is used, it may be desired to first convert the RNA to acomplementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to a selected nucleic acid sequence are contacted with thetemplate nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemiluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known (see Sambrook et al., 1989). Alternativemethods for reverse transcription utilize thermostable DNA polymerases.These methods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). European Application No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) discloses a nucleic acid sequence amplification scheme basedon the hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “race” and “one-sided PCR” (Frohman, 1990; Oharaet al.,1989).

2. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic DNA, cDNAand/or RNA samples. Methods used to detect point mutations includedenaturing gradient gel electrophoresis (“DGGE”), restriction fragmentlength polymorphism analysis (“RFLP”), chemical or enzymatic cleavagemethods, direct sequencing of target regions amplified by PCR™ (seeabove), single-strand conformation polymorphism analysis (“SSCP”) andother methods well known in the art.

One method of screening for point mutations is based on RNase cleavageof base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As usedherein, the term “mismatch” is defined as a region of one or moreunpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNAor DNA/DNA molecule. This definition thus includes mismatches due toinsertion/deletion mutations, as well as single or multiple base pointmutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. For the detection of mismatches, the single-stranded productsof the RNase A treatment, electrophoretically separated according tosize, are compared to similarly treated control duplexes. Samplescontaining smaller fragments (cleavage products) not seen in the controlduplex are scored as positive.

Other investigators have described the use of RNase I in mismatchassays. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches. Othershave described using the MutS protein or other DNA-repair enzymes fordetection of single-base mismatches.

Alternative methods for detection of deletion, insertion or substitutionmutations that may be used in the practice of the present invention aredisclosed in U.S. Pat. Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525and 5,928,870, each of which is incorporated herein by reference in itsentirety.

3. Kits

All the essential materials and/or reagents required for detecting atarget ligand may be provided by the invention and may be assembledtogether in a kit. This generally will comprise an antibody or bindingprotein prepared in accordance with the invention and designed to haveaffinity specifically to a target ligand. Also included may be buffersto provide the necessary mixture for binding to the ligand, as well aslabeling means for detecting the binding. Such kits may also includeenzymes and other reagents suitable for detection of specific ligands.Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent or enzyme as well as for antibodyor binding protein.

IX. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Fluorescence Detection and Enrichment of Cells Expressing scFvAntibodies in the Periplasm

The 26-10 scFv antibody binds with high affinity to cardiac glycosidessuch as digoxin and digoxigenin (K_(D) of the purified antibodies fordigoxin and digoxigenin are 0.9±0.2×10⁻¹ M⁻¹ and 2.4±0.4×10⁻⁹ M⁻¹,respectively, Chen et al., 1999). The 26-10 scFv and its variants havebeen used extensively as a model system to understand the effect ofmutations in the CDRs and in the framework regions on hapten binding(Schilbach et al., 1992; Short et al., 1995; Daugherty et al., 1998,2000; Chen et al., 1999). A derivative of the 26-10 scFv was expressedunder the E. coli arabinose promoter and with the pelB leader peptidethat allows secretion in the E. coli periplasm. The resulting plasmidvector (pBAD30pelB-Dig) was transformed in the ara E. coli strain LMG194and protein synthesis was induced with 0.2% w/v arabinose. It wasobserved that upon incubation with 200 nM of digoxigenin-BODIPY™, cellsthat had been grown at 25° C. became strongly fluorescent and thefluorescence signal was retained even after extensive washing to removenon-specifically bound ligand. The labeling of the cells with a probehaving a M.W. which is significantly higher than the generally acceptedsize limit of about 600 Da for the permeation of hydrophilic solutes inthe periplasm (Decad and Nikaido, 1976) raised the possibility that thefluorescence signal was mainly due to non-viable, permeabilized cells.However, staining with the viability stain propidium iodide, which bindsspecifically to membrane damaged cells by virtue of intercalating withthe normally inaccessible nucleic acids, revealed that >90% of the cellswere not permeable to the dye. This is similar to the proportion ofintact cells in control E. coli cultures harvested in late exponentialphase.

Cells expressing the 26-10 antibody in the periplasm could be enrichedfrom a large excess of E. coli transformed with vector alone in a singleround of sorting. Specifically, LMG194 (pBAD30pe1B-Dig) were mixed witha 10,000 fold excess of E. coli containing empty vector (pBAD30). Theformer cells are resistant to both ampicillin and chloramphenicol(amp^(r), Cm^(r)) whereas the latter are resistant to ampicillin only(amp^(r)). 4 hours after induction with 0.2% w/v arabinose, the cellswere then labeled with 100 nM digoxigenin-BODIPY™ for 1 hour andfluorescent cells were isolated by FACS. Following re-growth of thesorted cells and re-labeling as above, the population exhibited a fiveto eight-fold increase in the mean fluorescence intensity (FL1=20 vsFL1=4 for the pre-sort cell mixture). The fraction of scFv-expressingclones in the enriched population was estimated from the number of amprclones that were also Cm^(r). 80% of the ampr colonies were also Cm^(r)indicating that fluorescence labeling and cell sorting gave anenrichment of well over 1,000-fold in a single round

EXAMPLE 2 Antibody Affinity Maturation

Short et. al., (1995) isolated a 26-10 mutant, designated A4-19, havingan equilibrium dissociation constant (KD) for digoxin of 300 pM asmeasured by surface plasmon resonance. A4-19 contains 3 amino acidsubstitutions in heavy chain CDR1 (V_(H):T30->P, V_(H):D31->S and V_(H):M34->Y). It was examined whether mutants with increased bindingaffinity can be obtained by periplasmic expression/FACS screening evenwhen starting with an antibody that already exhibits very tight binding.Three light chain CDR3 residues that make contact (V_(L):T91, V_(L):P96)or are in close proximity to (V_(L):V94) the digoxin hapten (Jeffrey.etal., 1993) were randomized using an NNS (S=G or C) strategy (Daughertyet al. 1998). A library of 2.5×10⁶ transformants expressed in theperiplasm via the pelB leader was generated and screened using tworounds of FACS (FIG. 2). In the first round of screening, cells labeledwith 100 nM of the fluorescent probe were washed once with PBS andsorted using recovery mode in which the instrument collects allfluorescent events even if a non-fluorescent particle is detected in thesame element of fluid as a fluorescent particle. Operation in recoverymode provided a better assurance that very rare cells would be collectedbut at the expense of purity.

Collected cells were re-grown, labeled, washed and then incubated with a50-fold excess (50 μM) of free digoxin for various times (15 min to 90min). Cells that retained the desired level of fluorescence wereisolated by sorting using exclusion mode, in which, coincidentfluorescent and non-fluorescent events were rejected and thus a higherdegree of purity was obtained. The rate of fluorescence decay for thepool of cells obtained following incubation with non-fluorescentcompetitor for various times was measured. A slightly faster ratecompared to the starting A4-19 antibody was observed for the earliertime points (<60 minutes incubation with competitor) but the rate wasreduced for the 60 min and 90 min populations. 5 random clones from thecell population obtained after 60 min of competition and 13 clones fromthe 90 min pool were picked at random and sequenced (Table 1). A strongsequence consensus was clearly evident. The hapten binding kinetics ofthe purified antibodies were determined by SPR and the results are shownin Table 1. The corresponding amino acid sequences are given by SEQ IDNOs:1-12. It should be noted that upon purification and analysis by gelfiltration FPLC none of the mutants was found to dimerize. All of themutants examined displayed association rate constants (k_(on))indistinguishable from that of the starting A4-19 antibody (0.9±0.2×10⁶M⁻¹). The k_(diss) of the clones isolated after 60 min of competitionwere the same or faster than that of A4-19. Clones isolated after 90minutes of competition exhibited slower k_(diss) in solution. One clone,90.3, exhibited a 2-fold slower dissociation rate constant resulting ina K_(D) of 150 pM. Thus, the library screening methodology of theinvention allowed specific labeling to isolate a better mutant, evenwhen starting with an antibody that already exhibited a sub-nanomolarK_(D). Interestingly, but not surprisingly, the effect of the threeheavy chain CDR1 mutations present in 4-19 and the two mutations inresidues 94 and 96 of the light chain were additive.

TABLE 1 Heavy and light chain CDR3 amino acid sequences (SEQ ID NOs:1-12) of mutants isolated by 60 min (clones 60.1-60.4 and 90 minutes(clones 90.1-90.6) off-rate selection. Light Chain Sequence 90 . . . 96Off-rate/s Wild Type 26-10 scFv Q T T H V P P 8.4 × 10⁻⁴ A14-9 Q T T H VP P 2.7 × 10⁻⁴ 60.1 (1 clone) Q T T H S P A 5.5 × 10⁻⁴ 60.2 (2) Q T T HL P T 2.8 × 10⁻⁴ 60.3 (1) Q T T H T P P ND 60.4 (1) Q T T H L P A ND90.1 (1) Q T T H I P T 3.2 × 10⁻⁴ 90.2 (1) Q T T H V P P 2.7 × 10⁻⁴ 90.3(7) Q T T H V P A 2.2 × 10⁻⁴ 90.4 (1) Q T T H I P A 1.4 × 10⁻⁴ 90.5 (3)Q T T H L P A ND 90.6 (1) Q T T H V P C ND Number of identical clonesshown in parenthesis. ND: Not Determined.

EXAMPLE 3 Maximizing the Fluorescence Signal

The fluorescence intensity of cells expressing scFv antibodies insoluble form in the periplasm was strongly dependent on the E. colistrain used and on the growth conditions. With the 26-10 antibody, themaximum fluorescence intensity was obtained when the cells were grown at25° C. Growth at sub-physiological temperature has several beneficialeffects. Expression of scFv at low temperature (i.e., 25° C.)facilitates the proper folding of the scFv both directly, by slowing thefolding pathway and indirectly by decreasing plasmid copy number toreduce expression load. Indeed, direct expression of scFv at 37° C.generally yields little or no soluble protein (for example see Gough etal., 1999). Outer membrane composition is also altered atnon-physiological temperatures resulting in increased permeability(Martinez et al., 1999). Rather dramatic differences among various E.coli strains were noticed. Among several strains tested, the highestfluorescence intensities were obtained in ABLE™C (FIG. 3). A preliminaryanalysis of protein expression and outer membrane protein profile inthis strain indicated that the higher fluorescent signal was not due tothe pcnB mutation which reduces the copy number of ColE1 origin plasmidsbut rather, due to differences in cell envelope protein composition. Infact, the stronger staining of ABLE™C was not related to a higher levelof protein expression relative to other strains as deduced by ELISA andWestern blotting.

Fluorescent labeling under hyperosmotic conditions, resulted insignificantly greater fluorescence (FIG. 4). A 5-7 fold increase influorescence was obtained when the cells were incubated in 5× PBS duringlabeling (a mean FL1>150 compared to 20-30 for cells incubated inregular PBS). However, the increased signal came at a cost, as cellviability decreased considerably. Such a decrease in viability may beundesirable when screening highly diverse libraries of proteins, whoseexpression may already have a deleterious effect on the host cell.Similarly, co-infection with filamentous phages such as M13KO7 inducesthe phage shock response, which among other things, results in anincrease in outer membrane permeability. M13 KO7 infection resulted in a3-fold increase in the mean fluorescence of the population (FIG. 5).However, as with hyperosmotic shock the viability of the culture, asdetermined by propidium iodide staining was somewhat decreased.

Labeling of the cells with fluorescent ligand followed by incubationwith a large excess of free ligand results in a time-dependent decreasein the mean fluorescence intensity. The rate of the fluorescence decayreflects the dissociation rate of the antibody-antigen complex(Daugherty et al., 2000). For digoxin the rate of fluorescence decay wasfound to be about 3-4 times slower compared to the dissociation ratemeasured with the purified antibody using BIACORE. The lower rate offluorescence decay compared to the dissociation rate of theantibody/antigen complex in vitro stems from several effects includingthe collision frequency between ligands and cells, the concentration ofantibody in the periplasm and, of course, the rate of diffusion throughthe outer membrane (see Martinez et al., (1996) for an analysis ofkinetics in the periplasmic space). As may be expected, the ratio of therate of fluorescence decay in the periplasm relative to the in vitrodetermined k_(off) rate is antigen dependent.

EXAMPLE 4 Analysis and Screening of Repertoire Antibody Libraries byFACS

Antibodies can be isolated de novo, i.e., without animal immunization,by screening large, repertoire libraries that contain a wide variety ofantibody sequences. The screening of such large libraries is wellestablished (Nissim et al. 1994, Winter et al. 1994, Griffith et al.1994, Knappik et al. 2000). It was important to establish: (a) Howanchor-less display (ALD) compares with the phage display technology interms of allowing the isolation of high affinity clones and (b) whetherALD can be used to screen highly diverse libraries.

So far all the large antibody repertoire libraries available have beenconstructed for use with phage display. The inventors discovered thatthey could take advantage of the fact that libraries constructed forphage display also allow the expression of proteins within the bacterialperiplasmic space. In particular, for low protein copy number display onfilamentous bacteriophage, recombinant polypeptides are expressed asN-terminal fusions to pIII. During the course of phage biogenesis, pIIIfusions are first targeted to the periplasm and anchored in the innermembrane by a small C-terminal portion of pIII. As phage are released,the scFv-pIII fusion is incorporated alongside wild-type pIII at theterminus of the phage, thereby concluding the assembly process (Rakonjacand Model, 1998; Rakonjac et al., 1999). In the most widely used vectorsfor phage display an amber codon is placed between the N-terminal scFvand the pIII gene. Thus, in a suitable E. coli suppressor strain,full-length scFv-pIII fusion protein is produced for displaying the scFvwhereas in a non-supressor strain only soluble scFv is expressed. Thedegree of suppression varies with vector and strain but tends to allowonly 10% read-through. Thus, as a consequence of the biology of phagedisplay, all amber-codon containing libraries result in a degree ofperiplasmic expression regardless of host. Hence, it was of greatinterest to explore whether FACS can aid the isolation of ligand bindingproteins from pre-existing, highly diverse, naive libraries (Griffithset al., 1994; Vaughan et al., 1996; Sheets et al., 1998; Pini et al.,1998; de Haard et al., 1999; Knappik et al., 2000; Sblattero andBradbury, 2000).

Conventional screening of the phage library by phage panning enrichedphage expressing scFvs specific for the cardiac glycoside digoxin (FIG.6A, B) from a naïve antibody repertoire library. The panning process wasperformed on a BSA conjugate and the screening was performed on anovalbumin conjugate to reduce the incidence of protein andhapten-protein interface binders. 24 positive isolates from pan 4 sharedthe same fingerprint and DNA sequencing of 6 clones confirmed the sameheavy and light chain sequence (“dig1”) with one of six (“dig2”) havinga unique HCDR3 and LCDR3 combination (FIG. 7). Repeated screening of thephage library both under identical and under different conditionsresulted only in the isolation of clones with the same DNA fingerprint.

FACS analysis of the phage rescued in E. coli ABLE™C after each round ofpanning reveals an increase in mean fluorescence at round 3 whichmirrors the phage ELISA signals (FIG. 6C). Significant enrichment ofbinding clones using a single round of FACS was obtained starting withthe population obtained from the 3^(rd) round of phage panning. Thisresult is consistent with the enrichment profiles obtained during thecourse of the panning experiment. FACS screening and sorting 106 cellsfrom rounds 3, 4 and 5 resulted in the isolation of positive clones at afrequency of 30, 80 and 100% respectively.

Out of 14 clones isolated by FACS from the round 3 population 5 werefound to be positive for binding to digoxin. Importantly three of theclones corresponded to a different antibody that was missed by phagepanning (herein known as “dig3”). The remaining 2 were the dig1 clone.This result demonstrates that FACS screening of libraries expressed inthe periplasmic space and labeled with fluorescent ligands results inthe isolation of clones that cannot be isolated by other libraryscreening methodologies.

EXAMPLE 5 Materials And Methods

A. Strains and Plasmids

E. coli strains TG1 and HB2151 were provided with the Griffin library.ABLE™C and ABLE™K were purchased from Stratagene and helper phage M13K07from Pharmacia. A positive control for FACS analysis of a phage displayvehicle was constructed by replacing a pre-existing scFv in pHEN2 withthe 26.10 scFv to create pHEN2.dig. The negative control was pHEN2.thybearing the anti-thyroglobulin scFv provided with the Griffin.1 library.The P_(tac) vector was a derivative of pIMS120 (Hayhurst, 2000).

B. Phage Panning

The Griffin.1 library is a semi-synthetic scFv library derived from alarge repertoire of human heavy and light chains with part or all of theCDR3 loops randomly mutated and recombined in vivo (Griffiths et al.,1994). The library was rescued and subjected to five rounds of panningaccording to the web-site instruction manual (available on the worldwide web at mrc-cpe.cam.ac.uk/.about.phage/gl1p), summarized in Example6, below. Immunotubes were coated with 10 .mu.gml.sup.−1 digoxin-BSAconjugate and the neutralized eluates were halved and used to infecteither TG-1 for the next round of phage panning, or ABLE.TM. C for FACSanalysis.

Eluate titers were monitored to indicate enrichment of antigen bindingphage. To confirm reactivity, a polyclonal phage ELISA of purified,titer normalized phage stocks arising from each round was performed ondigoxin-ovalbumin conjugate. The percentage of positive clones arisingin rounds 3, 4 and 5 was established by monoclonal phage ELISA of 96isolates after each round. A positive was arbitrarily defined as anabsorbance greater than 0.5 with a background signal rarely above 0.01.MvaI fingerprinting was applied to 24 positive clones from rounds 3, 4and 5.

C. FACS screening

An aliquot of phagemid containing, ABLE™C glycerol stock was scrapedinto 1 ml of 2× TY (2% glucose, 100 μgml⁻ampicillin) to give an OD at600 nm of approximately 0.1 cm⁻¹. After shaking vigorously at 37° C. for2 h, IPTG was added to 1 mM and the culture shaken at 25° C. for 4 h. 50μl of culture was labeled with 100 nM BODIPY™-digoxigenin (Daugherty etal., 1999) in 1 ml of 5× PBS for 1 h at room temperature with moderateagitation. For the last 10 min of labeling, propidium iodide was addedto 2 μgml⁻¹. Cells were pelleted and resuspended in 100 μl of labelingmix. Scanning was performed with Becton-Dickinson FACSort, collecting10⁴ events at 1500 s⁻¹.

For FACS library sorting, the cells were grown in terrific broth andinduced with 0.1 mMIPTG. Sorting was performed on 10⁶ events (10⁷ forround 2) in exclusion mode at 1000 s⁻¹. Collected sort liquor was passedthrough 0.7 μm membrane filters and colonies allowed to grow afterplacing the filter on top of SOC agar plus appropriate antibiotics at30° C. for 24 h.

D. Analysis of phage clones

Screening phage particles by ELISA is summarized as follows. Binding ofphage in ELISA is detected by primary sheep anti-M13 antisera (CPlaboratories or 5 prime-3 prime) followed by a horseradish peroxidase(HRP) conjugated anti-sheep antibody (Sigma). Alternatively, aHRP-anti-M13 conjugate can be used (Pharmacia). Plates can be blockedwith 2% MPBS or 3% BSA-PBS. For the polyclonal phage ELISA, thetechnique is generally as follows: coat MicroTest III flexible assayplates (Falcon) with 100 μl per well of protein antigen. Antigen isnormally coated overnight at 4° C. at a concentration of 10-100 μg/ml ineither PBS or 50 mM sodium hydrogen carbonate, pH 9.6. Rinse wells 3times with PBS, by flipping over the ELISA plates to discard excessliquid, and fill well with 2% MPBS or 3% BSA-PBS for 2 hr at 37° C.Rinse wells 3 times with PBS. Add 10 μl PEG precipitated phage from thestored aliquot of phage from the end of each round of selection (about10¹⁰ tfu.). Make up to 100 μl with 2% MPBS or 3% BSA-PBS. Incubate for90 min at rt. Discard the test solution and wash three times withPBS-0.05% Tween 20, then 3 times with PBS. Add appropriate dilution ofHRP-anti-M13 or sheep anti-M13 antisera in 2% MPBS or 3% BSA-PBS.Incubate for 90 min at rt, and wash three times with PBS-0.05% Tween 20,then 3 times with PBS. If sheep anti-M13 antisera is used, incubate for90 min at rt, with a suitable dilution of P-anti-sheep antisera in 2%MPBS or 3% BSA and wash three times with PBS-0.05% Tween 20, then 3times with PBS. Develop with substrate solution (100 μg/ml TMB in 100 mMsodium acetate, pH 6.0, add 10 μl of 30% hydrogen peroxide per 50 ml ofthis solution directly before use). Add 100 μl to each well and leave atrt for 10 min. A blue color should develop. Stop the reaction by adding50 μl 1 M sulfuric acid. The color should turn yellow. Read the OD at450 nm and at 405 nm. Subtract OD 405 from OD 450.

Monoclonal phage ELISA can be summarized as follows. To identifymonoclonal phage antibodies the pHEN phage particles need to be rescued:Inoculate individual colonies from the plates in C10 (after each roundof selection) into 100 μl 2× TY containing 100 μg/ml ampicillin and 1%glucose in 96-well plates (Corning ‘Cell Wells’) and grow with shaking(300 rpm.) overnight at 30° C. Use a 96-well transfer device to transfera small inoculum (about 2 μl) from this plate to a second 96-well platecontaining 200 μl of 2× TY containing 100 μg/ml ampicillin and 1%glucose per well. Grow shaking at 37° C. for 1 hr. Make glycerol stocksof the original 96-well plate, by adding glycerol to a finalconcentration of 15%, and then storing the plates at −70° C. To eachwell (of the second plate) add VCS-M13 or M13KO7 helper phage to an moiof 10. Stand for 30 min at 37° C. Centrifuge at 1,800 g. for 10 min,then aspirate off the supernatant. Resuspend pellet in 200 μl 2× TYcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin. Grow shakingovernight at 30° C. Spin at 1,800 g for 10 min and use 100 μl of thesupernatant in phage ELISA as detailed above.

Production of soluble antibody fragments is summarized as follows: theselected pHEN needs to be infected into HB2151 and then induced to givesoluble expression of antibody fragments for ELISA. From each selectiontake 10 μl of eluted phage (about 10⁵ t.u.) and infect 200 μlexponentially growing HB2151 bacteria for 30 min at 37° C. (waterbath).Plate 1, 10, 100 μl, and 1:10 dilution on TYE containing 100 μg/mlampicillin and 1% glucose. Incubate these plates overnight at 37° C.Pick individual colonies into 100 μl 2× TY containing 100 μg/mlampicillin and 1% glucose in 96-well plates (Corning ‘Cell Wells’), andgrow with shaking (300 rpm.) overnight at 37° C. A glycerol stock can bemade of this plate, once it has been used to inoculate another plate, byadding glycerol to a final concentration of 15% and storing at −70° C.Use a 96-well transfer device to transfer a small inocula (about 2 μl)from this plate to a second 96-well plate containing 200 μl fresh 2× TYcontaining 100 μg/ml ampicillin and 0.1% glucose per well. Grow at 37°C., shaking until the OD at 600 nm is approximately 0.9 (about 3 hr).Once the required OD is reached add 25 μl 2× TY containing 100 μg/mlampicillin and 9 mM IPTG (final concentration 1 mM IPTG). Continueshaking at 30° C. for a further 16 to 24 hr. Coat MicroTest III flexibleassay plates (Falcon) with 100 μl per well of protein antigen. Antigenis normally coated overnight at rt at a concentration of 10-100 μg/ml ineither PBS or 50 mM sodium hydrogen carbonate, pH 9.6. The next dayrinse wells 3 times with PBS, by flipping over the ELISA plates todiscard excess liquid, and block with 200 μl per well of 3% BSA-PBS for2 hr at 37° C. Spin the bacterial plate at 1,800 g for 10 min and add100 μl of the supernatant (containing the soluble scFv) to the ELISAplate for 1 hr at rt. Discard the test solution and wash three timeswith PBS. Add 50 μl purified 9E10 antibody (which detects myc-taggedantibody fragments) at a concentration of 4 μg/ml in 1% BSA-PBS and 50μl of a 1:500 dilution of HRP-anti-mouse antibody in 1% BSA-PBS.Incubate for 60 min at rt, and wash three times with PBS-0.05% Tween 20,then 3 times with PBS. Develop with substrate solution (100 μg/ml TMB in100 mM sodium acetate, pH 6.0. Add 10 μl of 30% hydrogen peroxide per 50ml of this solution directly before use). Add 100 μl to each well andleave at rt for 10 min. A blue color should develop. Stop the reactionby adding 50 μl 1 M sulphuric acid. The color should turn yellow. Readthe OD at 450 nm and at 405 nm. Subtract OD 405 from OD 450.

Inserts in the library can be screened by PCR screening using theprimers designated LMB3: CAG GAA ACA GCT ATG AC (SEQ ID NO:13) and Fdseq1: GAA TTT TCT GTA TGA GG (SEQ ID NO:14). For sequencing of the VHand VL, use is recommend of the primers FOR_LinkSeq: GCC ACC TCC GCC TGAACC (SEQ ID NO:15) and pHEN-SEQ: CTA TGC GGC CCC ATT CA (SEQ ID NO:16).

EXAMPLE 6 Summary of Methodology for Use of the Griffin.1 Library

Methodology for using the Griffin.1 library can be summarized asfollows. The Griffin.1 library is a scFv phagemid library made fromsynthetic V-gene segments. The library was made by recloning the heavyand light chain variable regions from the lox library vectors (Griffithset al., EMBO J, 1994) into the phagemid vector pHEN2. A kit for use ofthe library will contain a tube of the synthetic scFv Library (1 ml), aglycerol stock of the positive control (TG1 containing ananti-thyroglobulin clone), a glycerol stock of the negative control (TG1containing pHEN2), a glycerol stock of E. coli TG1 (Gibson, 1984)suppressor strain (K12, del(lac-pro), supE, thi, hsdD5/F′traD36,proA+B+, lacIq, lacZdelM15) for propagation of phage particles (thestrain supplied is a T-phage resistant variant of this), a glycerolstock of E. coli HB2151 (Carter et al., 1985) and non-suppressor strain(K12, ara, del(lac-pro), thi/F′proA+B+, lacIq, lacZdelM15) forexpression of antibody fragments. The library is kept frozen at −70° C.until needed.

The strains are plated and then are grown up as overnight cultures(shaking at 37° C.) of each in 2× TY containing 100 μg/ml ampicillin and1% glucose. Cultures are diluted 1:100 with 2× TY (2× TY is 16 gTyptone, 10 g Yeast Extract and 5 g NaCl in 1 litre) containing 100μg/ml ampicillin and 1% glucose and the phagemids rescued by followingthe procedures described below. A 1:100 mixture is used of positive andthe negative control together for one round of selection on immunotubes,coated with thyroglobulin.

The protocol for use of the library is summarized as follows.Phage/phagemid infect F+-E. coli via the sex pili. For sex piliproduction and efficient infection E. coli must be grown at 37° C. andbe in log phase (OD at 600 nm of 0.4-0.6). Throughout the followingprotocol such a culture is needed. It can be prepared as follows:transfer a bacterial colony from a minimal media plate into 5 ml of 2×TY medium and grow shaking overnight at 37° C. Next day, subculture bydiluting 1:100 into fresh 2× TY medium, grow shaking at 37° C. until OD0.4-0.6 and then infect with phage. A variety of helper phages areavailable for the rescue of phagemid libraries. VCS-M13 (Stratagene) andM13KO7 (Pharmacia) can be purchased in small aliquots, larger quantitiesfor rescue of phagemid libraries can be prepared as follows: Infect 200μl E. coli TG1 (or other suitable strain) at OD 0.2 with 10 μl serialdilutions of helper phage (in order to get well separated plaques) at37° C. (waterbath) without shaking for 30 min. Add to 3 ml molten H-topagar (42° C.) and pour onto warm TYE (note 7) plates. Allow to set andthen incubate overnight at 37° C. Pick a small plaque into 3-4 ml of anexponentially growing culture of TG1 (see above). Grow for about 2 hrshaking at 37° C. Inoculate into 500 ml 2× TY in a 2 litre flask andgrow as before for 1 hr and then add kanamycin (25 μg/ml in water) to afinal concentration of 50-70 μg/ml. Grow for a further 8-16 hr. Spindown bacteria at 10,800 g for 15 min. To the phage supernatant add ⅕volume PEG/NaCl (20% polyethylene glycol 6000-2.5 M NaCl) and incubatefor a minimum of 30 min on ice. Spin 10,800 g for 15 min. Resuspendpellet in 2 ml TE and filter sterilise the stock through a 0.45 μnfilter (Minisart NML; Sartorius). Titre the stock and then dilute toabout 1×1012 p.f.u./ml. Store aliquots at −20° C. All spins areperformed at 4° C., unless otherwise stated.

For growth of the library, the procedure is summarized as follows:inoculate the whole of the bacterial library stock (about 1×10¹⁰ clones)into 500 ml 2× TY containing 100 μg/ml ampicillin and 1% glucose. Growwith shaking at 37° C. until the OD at 600 nm is 0.5, this should takeabout 1.5-2 hours. Infect 25 ml (1×1010 bacteria) from this culture withVCS-M13 or M13KO7 helper phage by adding helper phage in the ratio of1:20 (number of bacterial cells:helper phage particles, taking intoaccount that 1 OD bacteria at 600 nm=around 8×108 bacteria/ml).

Spin the infected cells at 3,300 g for 10 min. Resuspend the pelletgently in 30 ml of 2× TY containing 100 μg/ml ampicillin and 25 μg/mlkanamycin. Add 470 ml of prewarmed 2× TY containing 100 μg/ml ampicillinand 25 μg/ml kanamycin and incubate shaking at 30° C. overnight. Thephage can be concentrated and any soluble antibodies removed (as in TG1suppression of the amber stop codon encoded at the junction of theantibody gene and gIII is never complete) by precipitating withPolyethylene glycol (PEG) 6000. Spin the culture from A6 at 10,800 g for10 min (or 3,300 g for 30 min). Add ⅕ volume PEG/NaCl (20% Polyethyleneglycol 6000, 2.5 M NaCl) to the supernatant. Mix well and leave for 1 hror more at 4° C. Spin 10,800 g for 30 min. Resuspend the pellet in 40 mlwater and add 8 ml PEG/NaCl. Mix and leave for 20 min or more at 4° C.Spin at 10,800 g for 10 min or 3,300 g for 30 min and then aspirate offthe supernatant. Respin briefly and then aspirate off any remainingPEG/NaCl. Resuspend the pellet in 5 ml PBS and spin 11,600 g for 10 minin a microcentrifuge to remove most of the remaining bacterial debris.Store the phage supernatant at 4° C. for short term storage or in PBS,15% glycerol for longer term storage at −70° C. To titre the phage stockdilute 1 μl phage in 1 ml PBS and use 1 μl of this to infect 1 ml of TG1at an OD600 0.4-0.6. Plate 50 μl of this, 50 μl of a 1:102 dilution and50 μl of a 1:104 on TYE plates containing 100 μg/ml ampicillin and 1 %glucose and grow overnight at 37° C. Phage stock should be 10¹²-10¹³/ml.

Selection on immunotubes is summarized as follows. Coat Nunc-immunotube(Maxisorp Cat. No. 4-44202) overnight with 4 ml of the required antigen.The efficiency of coating can depend on the antigen concentration, thebuffer and the temperature. Usually 10-100 μg/ml antigen in PBS or 50 mMsodium hydrogen carbonate, pH 9.6 at room temperature (rt), is used.Next day wash tube 3 times with PBS (simply pour PBS into the tube andthen pour it immediately out again). Fill tube to brim with 2% MPBS.Cover and incubate at 37° C. (or rt according to the stability ofantigen) for 2 hr to block. Wash tube 3 times with PBS. Add 10¹² to 10¹³cfu. phage, from A13, in 4 ml of 2% MPBS. Incubate for 30 min at rtrotating continuously on an under-and-over turntable and then stand forat least a further 90 min at rt. Throw away the unbound phage in thesupernatant. For the first round of selection wash tubes 10 times withPBS containing 0.1% Tween-20, then 10 times with PBS to remove thedetergent. Each washing step is performed by pouring buffer in andimmediately out. For the second and subsequent rounds of selection washtubes 20 times with PBS containing 0.1% Tween-20, then 20 times withPBS. Shake out the excess PBS from the tube and elute phage by adding 1ml 100 mM triethylamine (700 μl triethylamine (7.18 M) in 50 ml water,diluted on day of use) and rotating continuously for 10 min on anunder-and-over turntable. During the incubation, tubes are prepared with0.5 ml 1M Tris, pH 7.4 ready to add the eluted 1 ml phage, from 7, forquick neutralisation. Phage can be stored at 4° C. or used to infect TG1as described above. After elution add another 200 μl of 1M Tris, pH 7.4to the immunotube to neutralise the remaining phage in the tube. Take9.25 ml of an exponentially growing culture of TG1 and add 0.75 ml ofthe eluted phage. Also add 4 ml of the TG1 culture to the immunotube.Incubate both cultures for 30 min at 37° C. (waterbath) without shakingto allow for infection. Pool the 10 ml and 4 ml of the infected TG1bacteria and take 100 μl to make 4-5 100-fold serial dilutions. Platethese dilutions on TYE containing 100 μg/ml ampicillin and 1% glucose.Grow overnight at 37° C. Take the remaining infected TG1 culture andspin at 3,300 g for 10 min. Resuspend the pelleted bacteria in 1 ml of2× TY and plate on a large Nunc Bio-Assay dish (Gibco-BRL (note 8)) ofTYE containing 100 μg/ml ampicillin and 1% glucose. Grow at 30° C.overnight, or until colonies are visible.

For further rounds of selection, add 5-6 ml of 2× TY, 15% glycerol tothe Bio-Assay dish of cells and loosen the cells with a glass spreader.After inoculating 50-100 μl of the scraped bacteria to 100 ml of 2× TYcontaining 100 μg/ml ampicillin and 1% glucose, store the remainingbacteria at −70° C. Once again it is a good idea to check starting OD at600 nm is=<0.1. Grow the bacteria with shaking at 37° C. until the OD at600 nm is 0.5 (about 2 hr). Infect 10 ml of this culture with VCS-M13 orM13KO7 helper phage by adding helper phage in the ratio of 1:20 (numberof bacterial cells:helper phage particles, taking into account that 1 ODbacteria at 600 nm=around 8×108 bacteria/ml). Incubate without shakingin a 37° C. water bath for 30 min. Spin the infected cells at 3,300 gfor 10 min. Resuspend the pellet gently in 50 ml of 2× TY containing 100μg/ml ampicillin and 25 μg/ml kanamycin and incubate shaking at 30° C.overnight. Take 40 ml of the overnight culture and spin at 10,800 g for10 min or 3,300 g for 30 min. Add ⅕ volume (8 ml) PEG/NaCl (20%Polyethylene glycol 6000, 2.5 M NaCl) to the supernatant. Mix well andleave for 1 hr or more at 4° C. Spin 10,800 g for 10 min or 3,300 g for30 min and then aspirate off the supernatant. Respin briefly and thenaspirate off any remaining dregs of PEG/NaCl. Resuspend the pellet in 2ml PBS and spin 11,600 g for 10 min in a micro centrifuge to remove mostof the remaining bacterial debris. 1 ml of this phage can be stored at4° C. and the other 1 ml aliquot can be used for the next round ofselection. Repeat the selection for another 2-3 rounds.

Screening phage particles by ELISA is summarized as follows. Binding ofphage in ELISA is detected by primary sheep anti-M13 antisera (CPlaboratories or 5 prime-3 prime) followed by a horseradish peroxidase(HRP) conjugated anti-sheep antibody (Sigma). Alternatively, aHRP-anti-M13 conjugate can be used (Pharmacia). Plates can be blockedwith 2% MPBS or 3% BSA-PBS. For the polyclonal phage ELISA, thetechnique is generally as follows: coat MicroTest III flexible assayplates (Falcon) with 100 μl per well of protein antigen. Antigen isnormally coated overnight at rt at a concentration of 10-100 μg/ml ineither PBS or 50 mM sodium hydrogen carbonate, pH 9.6. Rinse wells 3times with PBS, by flipping over the ELISA plates to discard excessliquid, and block with 200 μl per well of 2% MPBS or 3% BSA-PBS for 2 hrat 37° C. Rinse wells 3 times with PBS. Add 10 μl PEG precipitated phagefrom the stored aliquot of phage from the end of each round of selection(about 10¹⁰ cfu.). Make up to 100 μl with 2% MPBS or 3% BSA-PBS.Incubate for 90 min at rt. Discard the test solution and wash threetimes with PBS-0.05% Tween 20, then 3 times with PBS. Add appropriatedilution of HRP-anti-M13 or sheep anti-M13 antisera in 2% MPBS or 3%BSA-PBS. Incubate for 90 min at rt, and wash three times with PBS-0.05%Tween 20, then 3 times with PBS. If sheep anti-M13 antisera is used,incubate for 90 min at rt, with a suitable dilution of HRP-anti-sheepantisera in 2% MPBS or 3% BSA and wash three times with PBS-0.05% Tween20, then 3 times with PBS. Develop with substrate solution (100 μg/mlTMB in 100 mM sodium acetate, pH 6.0. Add 10 μl of 30% hydrogen peroxideper 50 ml of this solution directly before use). Add 100 μl to each welland leave at rt for 10 min. A blue colour should develop. Stop thereaction by adding 50 μl 1 M sulphuric acid. The colour should turnyellow. Read the OD at 450 nm and at 405 nm. Subtract OD 405 from OD450.

Monoclonal phage ELISA can be summarized as follows. To identifymonoclonal phage antibodies the pHEN phage particles need to be rescued:Inoculate individual colonies from the plates in C10 (after each roundof selection) into 100 μl 2× TY containing 100 μg/ml ampicillin and 1%glucose in 96-well plates (Corning ‘Cell Wells’) and grow with shaking(300 rpm.) overnight at 37° C. Use a 96-well transfer device to transfera small inoculum (about 2 μl) from this plate to a second 96-well platecontaining 200 μl of 2× TY containing 100 μg/ml ampicillin and 1%glucose per well. Grow shaking at 37° C. for 1 hr. Make glycerol stocksof the original 96-well plate, by adding glycerol to a finalconcentration of 15%, and then storing the plates at −70° C. To eachwell (of the second plate) add 25 μl 2× TY containing 100 μg/mlampicillin, 1% glucose and 109 pfu VCS-M13 or M13KO7 helper phage. Standfor 30 min at 37° C., then shake for 1 hr at 37° C. Spin 1,800 g. for 10min, then aspirate off the supernatant. Resuspend pellet in 200 μl 2× TYcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin. Grow shakingovernight at 30° C. Spin at 1,800 g for 10 min and use 100 μl of thesupernatant in phage ELISA as detailed above.

Production of soluble antibody fragments is summarized as follows: theselected pHEN needs to be infected into HB2151 and then induced to givesoluble expression of antibody fragments for ELISA. From each selectiontake 10 μl of eluted phage (about 105 t.u.) and infect 200 μlexponentially growing HB2151 bacteria for 30 min at 37° C. (waterbath).Plate 1, 10, 100 μl, and 1:10 dilution on TYE containing 100 μg/mlampicillin and 1% glucose. Incubate these plates overnight at 37° C.Pick individual colonies into 100 μl 2× TY containing 100 μg/mlampicillin and 1% glucose in 96-well plates (Corning ‘Cell Wells’), andgrow with shaking (300 rpm.) overnight at 37° C. A glycerol stock can bemade of this plate, once it has been used to inoculate another plate, byadding glycerol to a final concentration of 15% and storing at −70° C.Use a 96-well transfer device to transfer a small inocula (about 2 μl)from this plate to a second 96-well plate containing 200 μl fresh 2× TYcontaining 100 μg/ml ampicillin and 0.1% glucose per well. Grow at 37°C., shaking until the OD at 600 nm is approximately 0.9 (about 3 hr).Once the required OD is reached add 25 μl 2× TY containing 100 μg/mlampicillin and 9 mM IPTG (final concentration 1 mM IPTG). Continueshaking at 30° C. for a further 16 to 24 hr. Coat MicroTest III flexibleassay plates (Falcon) with 100 μl per well of protein antigen. Antigenis normally coated overnight at rt at a concentration of 10-100 μg/ml ineither PBS or 50 mM sodium hydrogen carbonate, pH 9.6. The next dayrinse wells 3 times with PBS, by flipping over the ELISA plates todiscard excess liquid, and block with 200 μl per well of 3% BSA-PBS. for2 hr at 37° C. Spin the bacterial plate at 1,800 g for 10 min and add100 μl of the supernatant (containing the soluble scFv) to the ELISAplate for 1 hr at rt. Discard the test solution and wash three timeswith PBS. Add 50 μl purified 9E10 antibody (which detects myc-taggedantibody fragments) at a concentration of 4 μg/ml in 1% BSA-PBS and 50μl of a 1:500 dilution of HRP-anti-mouse antibody in 1% BSA-PBS.Incubate for 60 min at rt, and wash three times with PBS-0.05% Tween 20,then 3 times with PBS. Develop with substrate solution (100 μg/ml TMB in100 mM sodium acetate, pH 6.0. Add 10 μl of 30% hydrogen peroxide per 50ml of this solution directly before use). Add 100 μl to each well andleave at rt for 10 min. A blue colour should develop. Stop the reactionby adding 50 μl 1 M sulphuric acid. The colour should turn yellow. Readthe OD at 450 nm and at 405 nm. Subtract OD 405 from OD 450.

Inserts in the library can be screened by PCR screening using theprimers designated LMB3: CAG GAA ACA GCT ATG AC (SEQ ID NO:13) and Fdseq1: GAA TTT TCT GTA TGA GG (SEQ ID NO:14). For sequencing of the VHand VL, use is recommend of the primers FOR_LinkSeq: GCC ACC TCC GCC TGAACC (SEQ ID NO:15) and pHEN-SEQ: CTA TGC GGC CCC ATT CA (SEQ ID NO:16).

EXAMPLE 7 Isolation of scFV Antibodies Specific to TNB from a RepertoireLibrary

This example summarizes the screening of a repertoire antibody libraryto the ligand TNB (trinitrobenzene). Library screening was initiated byfirst carrying out three rounds of phage panning of a repertoire library(Griffin-1 library) using standard protocols (see example 6, alsodescribed in available on the world wide web at mrc-cpe.cam.ac.u/ld.about.phage/glp). Phage rescued from various rounds ofpanning were used to infect the E. coli ABLE C. The cells were grown tomid-exponential phase, induced for expression of scFv antibodies asdescribed above and labeled with 100 nM TNBS conjugated to thefluorescent dye Cy5. The labeled cells were analyzed by flow cytometryusing a Cytomation MoFlo instrument equipped with a 5 mM diode laseremitting at 633 nm. Highly fluorescent clones were isolated on membranefilters and analyzed further. Three out of 10 clones isolated by FACSwere analyzed further and found to exhibit strong binding to a TNBS-BSAconjugate. Sequence analysis confirmed that one of the TNBS specificclones had also been found by phage display. However, the two otherclones isolated by the present invention (periplasmic expression of thelibrary and FACS screening) did not correspond to any of the clonesisolated by phage panning.

EXAMPLE 8 Detection of Oligonucleotide Probes by Antibodies Expressed inthe E. coli Periplasm

This example shows that modified oligonucleotides can diffuse throughthe outer membrane of bacteria. An oligonucleotide with the sequence5′-digoxigenin-AAAAA-fluoroscein-3′ (designated dig-5A-FL, molecularweight of 2,384 Da, SEQ ID NO:22) containing four nuclease resistantphosphorothioate linkages between the five A residues was synthesizedand purified (RP HPLC) by Integrated DNA Technologies, IA. Thedigoxigenin moiety of this oligonucleotide can be recognized by scFvantibodies specific to digoxin (anti-digoxin scFv). Cells expressing theanti-digoxin scFv in the periplasm may bind 5A-Fl which in turn shouldrender the cells fluorescent, provided that the probe molecule candiffuse through the outer membrane.

ABLE™C cells expressing periplasmic scFv specific for either atrazine(Hayhurst 2000) as a negative control (FIG. 8, panels i and iii) ordigoxigenin (FIG. 8, panels ii and iv) were incubated in 5× strength PBS(see example 3) together with either 100 nM of digoxigenin-BODIPY™ or100 nM of dig-5A-FL. Propidium iodine was also added to serve as aviability stain. Viable cells were gated on the basis of propidiumiodine exclusion (to identify cells with an intact membrane) and sidescatter. Approximately 10,000 cells were analyzed at a rate of 1,000events per second. The resulting data are shown in FIG. 8. Cellsexpressing an unrelated anti-atrazine antibody that does not bind to theprobe exhibited only background fluorescence. In contrast, cellsdisplaying the anti-digoxin scFv antibody became clearly labeled withboth the digoxigenin-BODIPY™ as well as with 5-A-FL. The latter probegave a signal that was clearly higher than that observed with thecontrol cells. Even though 5-A-FL gave a lower fluorescence intensitycompared to the smaller and uncharged the digoxigenin-BODIPY, the signalobtained with the former probe was sufficient for the screening of scFvlibraries by FACS.

EXAMPLE 9 Flow Cytometric Discrimination of E. coli Expressing theFusarium solani Lipase Cutinase Using Commercial Fluorescent Substrates

This example demonstrates that commercially available fluorescentsubstrates can be used to specifically label E. coli cells displayingthe relevant enzymes in their periplasm. Surprisingly, the fluorescentproduct of these reactions is sufficiently retained within the cell toallow for the discrimination and selection of enzyme expressing E. colifrom non-enzyme expressing bacteria.

The gene encoding Fusarium solani lipase cutinase was constructed bytotal gene synthesis and placed downstream of the strong induciblepromoter pBAD in plasmid pBAD18Cm. Protein expression from the pBADpromoter is beneficial for the screening of protein libraries by FACS(Daugherty et al. 1999). The resulting plasmid encoding the cutinasegene was designated pKG3-53-1. pKG3-53-1, and pBAD18Cm as a control,were both transformed into DH5a. In this example, the ability todiscriminate cells expressing cutinase (DH5a(pKG3-53-1)) from controlcells was determined using two different commercially availablesubstrates: Fluorescein dibutyrate or LysoSensor Green DND-189 (LSG)(both from Molecular Probes, OR). The latter is a positively chargedfluorescent probe that detects pH changes in the cell occurring due toester hydrolysis by the enzyme.

Cells were grown overnight with vigorous shaking at 37° C. in terrificbroth/chloramphenicol 50 μg/ml (TB/Cm). Subcultures were made from 100μl of overnight culture in 10 ml of TB/Cm(50 μg/ml). These subcultureswere grown with vigorous shaking at 37° C. to OD₆₀₀=0.6. Four mlaliquots of the subcultures were pelleted at 3650 rpm for 20 minutes ina Beckman Allegra 6R Centrifuge. The supernatant was removed, and thepellets were resuspended in 4 ml of M9 minimal media containing 0.2%glucose and chloramphenicol (Cm) at 50 μg/ml. Arabinose, from a 20%stock, was added to a final concentration of 0.2%. The cultures wereinduced at 25° C. with vigorous shaking for 4 hours. Subsequently, 2 mlaliquots of the induced cultures were pelleted at 8000 rpm for 10minutes in an Eppendorf 5415C Centrifuge, washed with fresh media andpelleted again at 8000 rpm for 10 min. The washed pellets wereresuspended in M9 salts media without glucose to an optical densityOD₆₀₀=1.0. The stock solution was diluted 1:10 and 1 ml of the dilutedcell suspension was mixed with 0.1 ml 0.1 mM Fluorescein dibutyrate(FDB) stock solution in dimethyl sulfoxide (DMSO). The final FDBconcentration was 10 μM. Reactions were allowed to proceed at 37° C. for30 minutes. The labeled cells were immediately analyzed on a BectonDickinson FACSort equipped with an Ar 488 nm laser. The fluorescencedistribution of the cutinase expressing cells and the control cells isshown in FIG. 9A.

The utility of a second probe for the discrimination between positive(enzyme expressing) and control cells was also examined. E. coliexpressing cutinase from the pKG3-53-4 plasmid, and negative cells(expressing the unmodified pBAD18Cm plasmid) were grown, induced andwashed as above. The pellet was washed with 1% sucrose, pelleted again,and resuspended in fresh 1% sucrose to OD₆₀₀=1.0. This stock solution ofcells was kept on ice.

For labeling, a LysoSensor Green DND-189 (LSG, Molecular Probes) stocksolution was prepared to 1 mM in DMSO. Also, a 1 M 4-NitrophenylButyrate stock solution was prepared in DMSO. Cell labeling wasinitiated by first diluting the cell stock solution, adding the LSG to afinal concentration of 1 μM and diluting the 4-Nitrophenyl Butyrate1:1000 to give a final concentration of 1 μM. The enzymatic hydrolysisof 4-Nitrophenyl Butyrate by the cells was allowed to proceed at 25° C.for 5 minutes and the cells were then immediately analyzed on a BectonDickinson FACSort as above. The fluorescence distribution of thecutinase expressing cells and the control cells stained with theLysoSensor Green DND-189 probe is shown in FIG. 9B.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of obtaining a bacterium comprising a nucleic acid sequenceencoding a catalytic protein catalyzing a chemical reaction involving atarget substrate, the method comprising the steps of: (a) providing aGram negative bacterium comprising a nucleic acid sequence encoding aheterologous candidate catalytic protein, wherein said catalytic proteinis expressed in soluble form in the periplasm of said bacterium; (b)contacting said bacterium with a target substrate capable of diffusinginto said bacterium, wherein said target substrate comprises a moleculecontaining a scissile amide bond, a scissile carboxylic ester bond, ascissile phosphate ester bond, a scissile sulfonate ester bond, ascissile carbonate ester bond, a scissile carbamate bond, a scissilethioester bond, a nucleic acid, or a polypeptide and said candidatecatalytic protein catalyzes a chemical reaction involving said targetsubstrate and wherein said chemical reaction yields at least a firstsubstrate product; and (c) selecting said bacterium based on thepresence of said first substrate product.
 2. The method of claim 1,further defined as a method of obtaining a nucleic acid sequenceencoding a catalytic protein catalyzing a reaction with a targetsubstrate, the method further comprising the step of: (d) cloning saidnucleic acid sequence encoding said candidate catalytic protein.
 3. Themethod of claim 1, wherein said nucleic acid sequence encoding acandidate catalytic protein is further defined as operably linked to aleader sequence capable of directing expression of said candidatecatalytic protein in said periplasm.
 4. The method of claim 1, whereinsaid Gram negative bacterium is an E. coli bacterium.
 5. The method ofclaim 1, further defined as comprising providing a population of Gramnegative bacteria.
 6. The method of claim 5, wherein said population ofbacteria is further defined as collectively capable of expressing aplurality of candidate catalytic proteins.
 7. The method of claim 6,wherein said population of bacteria is obtained by a method comprisingthe steps of: (a) preparing a plurality DNA inserts which collectivelyencode a plurality of different candidate catalytic proteins, and (b)transforming a population of Gram negative bacteria with said DNAinserts.
 8. The method of claim 5, wherein said population of Gramnegative bacteria is contacted with said target substrate.
 9. The methodof claim 1, wherein said candidate catalytic protein is further definedas an enzyme.
 10. The method of claim 1, wherein said candidatecatalytic protein is further defined as not capable of diffusing out ofsaid periplasm.
 11. The method of claim 1, wherein said target substratecomprises a molecule containing a scissile amide bond.
 12. The method ofclaim 1, wherein said target substrate comprises a polypeptide.
 13. Themethod of claim 1, wherein said target substrate comprises a moleculecontaining a scissile carboxylic ester bond.
 14. The method of claim 1,wherein said target substrate comprises a nucleic acid.
 15. The methodof claim 1, wherein said target substrate comprises a moleculecontaining a scissile phosphate ester bond.
 16. The method of claim 1,wherein said target substrate comprises a molecule containing a scissilesulfonate ester bond.
 17. The method of claim 1, wherein said targetsubstrate comprises a molecule containing a scissile carbonate esterbond.
 18. The method of claim 1, wherein said target substrate comprisesa molecule containing a scissile carbamate bond.
 19. The method of claim1, wherein said target substrate comprises a molecule containing ascissile thioester bond.
 20. The method of claim 1, wherein said targetsubstrate is further defined as comprising a molecular weight of lessthan about 20,000 Da.
 21. The method of claim 1, wherein said targetsubstrate is further defined as comprising a molecular weight of lessthan about 5,000 Da.
 22. The method of claim 1, wherein said targetsubstrate is further defined as comprising a molecular weight of lessthan about 3,000 Da.
 23. The method of claim 1, wherein said targetsubstrate is further defined as comprising a molecular weight of greaterthan about 600 Da and less than about 30,000 Da.
 24. The method of claim1, wherein said first substrate product is further defined as capable ofbeing detected based on the presence of a fluorescent signature.
 25. Themethod of claim 24, wherein said fluorescent signature is absent in saidtarget substrate.
 26. The method of claim 25, wherein said fluorescentsignature is produced by catalytic cleavage of a scissile bond.
 27. Themethod of claim 26, further defined as comprising use of a FRET system,said FRET system comprising a fluorophore bound by a scissile bond to atleast a first molecule capable of quenching the fluorescence of saidfluorophore, wherein cleavage of said scissile bond allows said firstmolecule to diffuse away from the fluorophore and wherein thefluorescence of said fluorophore becomes detectable.
 28. The method ofclaim 27, wherein the fluorophore comprises a positive charge allowingthe fluorophore to remain associated with the bacterium.
 29. The methodof claim 24, wherein said target substrate is further defined ascomprising a latent fluorescent moiety capable of being released by saidchemical reaction involving said target substrate.
 30. The method ofclaim 29, wherein the latent fluorescent moiety released by saidcleavage possesses an overall positive charge allowing said moiety toremain associated with the bacterium following said cleavage.
 31. Themethod of claim 24, further defined as comprising labeling said targetsubstrate with a fluorescent pH probe capable of being detected upon achange in pH associated with said chemical reaction involving saidtarget substrate.
 32. The method of claim 31, wherein said fluorescentpH probe possesses an overall positive charge allowing said fluorescentpH probe to remain associated with the bacterium following said chemicalreaction involving said target substrate.
 33. The method of claim 1,wherein said bacterium is further defined as viable following saidselecting.
 34. The method of claim 1, further comprising treating saidbacterium to facilitate said diffusing into said periplasm.
 35. Themethod of claim 34, comprising treating the bacterium with hyperosmoticconditions.
 36. The method of claim 34, comprising treating thebacterium with physical stress.
 37. The method of claim 34, comprisingtreating the bacterium with a phage.
 38. The method of claim 1, whereinsaid bacterium is grown at a sub-physiological temperature.
 39. Themethod of claim 38, wherein said sub-physiological temperature is about25° C.
 40. The method of claim 1, wherein said selecting comprises FACS.41. The method of claim 1, wherein said selecting comprises magneticseparation.